Compositions and methods for modulating bdnf expression

ABSTRACT

Aspects of the invention provide single stranded oligonucleotides for activating or enhancing expression of BDNF. Further aspects provide compositions and kits comprising single stranded oligonucleotides for activating or enhancing expression of BDNF. Methods for modulating expression of BDNF using the single stranded oligonucleotides are also provided. Further aspects of the invention provide methods for selecting a candidate oligonucleotide for activating or enhancing expression of BDNF.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/648,058, entitled “COMPOSITIONS ANDMETHODS FOR MODULATING BDNF EXPRESSION”, filed May 16, 2012, which isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to oligonucleotide based compositions, as well asmethods of using oligonucleotide based compositions for treatingdisease.

BACKGROUND OF THE INVENTION

Neurodegeneration is the progressive loss of function or death ofneurons and is the cause of a family of devastating diseases includingamyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease),Alzheimer's Disease (AD), Huntington's disease (HD) and Parkinson'sDisease (PD).

ALS involves degeneration of motor neurons and results in progressivemuscle weakness, dysarthria, dysphagia, respiratory difficulty, andeventually death. ALS can be caused by mutations in Cu/Zn superoxidedismutase 1. AD involves degeneration of neurons and synapses in thecerebral cortex, resulting in dementia, confusion, aggression, andlong-term memory loss. AD is hypothesized to be caused by misfoldedproteins that form small plaques that cause neuronal death. PD involvesthe death of dopamine-generating neurons in the substantia nigra,resulting in motor defects, psychiatric problems, and autonomicdysfunction. Mutations in some genes, alpha-synuclein (SNCA), parkin(PRKN), leucine-rich repeat kinase 2 (LRRK2 or dardarin), PTEN-inducedputative kinase 1 (PINK1), DJ-1 and ATP13A2, cause at least a subset ofParkinson's disease.

Brain-derived neurotrophic factor (BDNF) is a secreted neurotrophininvolved in neuron survival, growth, and differentiation. Additionally,BDNF is important in synaptic transmission and synapse plasticity.Several studies link BDNF with neurodegenerative diseases. BDNFexpression is reduced in patients with neurodegenerative diseases suchas Alzheimer's disease. Animal studies of neurodegeneration have shownthat elevated levels of BDNF can alleviate or are associated withalleviation of the symptoms of AD and PD. Additionally, BDNF mimeticsreduced neurodegneration in animal models of AD.

SUMMARY OF THE INVENTION

Aspects of the invention disclosed herein provide methods andcompositions that are useful for upregulating BDNF in cells. In someembodiments, single stranded oligonucleotides are provided that target aPRC2-associated region of a BDNF gene (e.g., human BDNF) and therebycause upregulation of the gene. In some embodiments, single strandedoligonucleotides are provided that target a PRC2-associated region ofthe gene encoding BDNF. In some embodiments, these single strandedoligonucleotides activate or enhance expression of BDNF by relieving orpreventing PRC2 mediated repression of BDNF. Aspects of the inventiondisclosed herein provide methods and compositions that are useful forupregulating BDNF for the treatment and/or prevention ofneurodegeneration diseases, such as amyotrophic lateral sclerosis (ALS,also known as Lou Gehrig's disease), AD, Huntington's disease (HD), orParkinson's Disease (PD).

Further aspects of the invention provide methods for selectingoligonucleotides for activating or enhancing expression of BDNF. In someembodiments, methods are provided for selecting a set ofoligonucleotides that is enriched in candidates (e.g., compared with arandom selection of oligonucleotides) for activating or enhancingexpression of BDNF. Accordingly, the methods may be used to establishsets of clinical candidates that are enriched in oligonucleotides thatactivate or enhance expression of BDNF. Such libraries may be utilized,for example, to identify lead oligonucleotides for developingtherapeutics to treat BDNF. Furthermore, in some embodiments,oligonucleotide chemistries are provided that are useful for controllingthe pharmacokinetics, biodistribution, bioavailability and/or efficacyof the single stranded oligonucleotides for activating expression ofBDNF.

According to some aspects of the invention single strandedoligonucleotides are provided that have a region of complementarity thatis complementarty with (e.g., at least 8 consecutive nucleotides of) aPRC2-associated region of a BDNF gene, e.g., a PRC2-associated region ofthe nucleotide sequence set forth as SEQ ID NO: 1 or 2. In someembodiments, the oligonucleotide has at least one of the followingfeatures: a) a sequence that is 5′X—Y—Z, in which X is any nucleotideand in which X is at the 5′ end of the oligonucleotide, Y is anucleotide sequence of 6 nucleotides in length that is not a human seedsequence of a microRNA, and Z is a nucleotide sequence of 1 to 23nucleotides in length; b) a sequence that does not comprise three ormore consecutive guanosine nucleotides; c) a sequence that has less thana threshold level of sequence identity with every sequence ofnucleotides, of equivalent length to the second nucleotide sequence,that are between 50 kilobases upstream of a 5′-end of an off-target geneand 50 kilobases downstream of a 3′-end of the off-target gene; d) asequence that is complementary to a PRC2-associated region that encodesan RNA that forms a secondary structure comprising at least two singlestranded loops; and e) a sequence that has greater than 60% G-C content.In some embodiments, the single stranded oligonucleotide has at leasttwo of features a), b), c), d), and e), each independently selected. Insome embodiments, the single stranded oligonucleotide has at least threeof features a), b), c), d), and e), each independently selected. In someembodiments, the single stranded oligonucleotide has at least four offeatures a), b), c), d), and e), each independently selected. In someembodiments, the single stranded oligonucleotide has each of featuresa), b), c), d), and e). In certain embodiments, the oligonucleotide hasthe sequence 5′X—Y—Z, in which the oligonucleotide is 8-50 nucleotidesin length.

According to some aspects of the invention, single strandedoligonucleotides are provided that have a sequence X—Y—Z, in which X isany nucleotide, Y is a nucleotide sequence of 6 nucleotides in lengththat is not a seed sequence of a human microRNA, and Z is a nucleotidesequence of 1 to 23 nucleotides in length, in which the single strandedoligonucleotide is complementary with a PRC2-associated region of a BDNFgene, e.g., a PRC2-associated region of the nucleotide sequence setforth as SEQ ID NO: 1 or 2. In some aspects of the invention, singlestranded oligonucleotides are provided that have a sequence 5′-X—Y—Z, inwhich X is any nucleotide, Y is a nucleotide sequence of 6 nucleotidesin length that is not a seed sequence of a human microRNA, and Z is anucleotide sequence of 1 to 23 nucleotides in length, in which thesingle stranded oligonucleotide is complementary with at least 8consecutive nucleotides of a PRC2-associated region of a BDNF gene,e.g., a PRC2-associated region of the nucleotide sequence set forth asSEQ ID NO: 1 or 2. In some embodiments, Y is a sequence selected fromTable 1. In some embodiments, the PRC2-associated region is a sequencelisted in any one of SEQ ID NOS: 7 to 68.

In some embodiments, the single stranded oligonucleotide comprises anucleotide sequence as set forth in any one of SEQ ID NOS: 69 to 62225,or a fragment thereof that is at least 8 nucleotides. In someembodiments, the single stranded oligonucleotide comprises a nucleotidesequence as set forth in any one of SEQ ID NOS: 69 to 62225, in whichthe 5′ end of the nucleotide sequence provided is the 5′ end of theoligonucleotide. In some embodiments, the region of complementarity(e.g., the at least 8 consecutive nucleotides) is also present withinthe nucleotide sequence set forth as SEQ ID NO: 5 or 6.

In some embodiments, the single stranded oligonucleotide comprises anucleotide sequence as set forth in any one of SEQ ID NOS: 69 to 62225.In some embodiments, the single stranded oligonucleotide comprises afragment of at least 8 nucleotides of a nucleotide sequence as set forthin any one of SEQ ID NOS: 69 to 62225.

In some embodiments, the PRC2-associated region is a sequence listed inany one of SEQ ID NOS: 7 to 48. In some embodiments, the single strandedoligonucleotide comprises a nucleotide sequence as set forth in any oneof SEQ ID NOS: 69 to 30107 or 62210 to 62225 or a fragment thereof thatis at least 8 nucleotides. In some embodiments, the single strandedoligonucleotide comprises a nucleotide sequence as set forth in any oneof SEQ ID NOS: 69 to 30107 or 62210 to 62225, wherein the 5′ end of thenucleotide sequence provided in any one of SEQ ID NOS: 69 to 30107 or62210 to 62225 is the 5′ end of the oligonucleotide. In someembodiments, the at least 8 consecutive nucleotides are also presentwithin the nucleotide sequence set forth as SEQ ID NO: 5.

In some embodiments, the PRC2-associated region is a sequence listed inany one of SEQ ID NOS: 49 to 68. In some embodiments, the singlestranded oligonucleotide comprises a nucleotide sequence as set forth inany one of SEQ ID NOS: 30003 to 62209 or a fragment thereof that is atleast 8 nucleotides. In some embodiments, the single strandedoligonucleotide comprises a nucleotide sequence as set forth in any oneof SEQ ID NOS: 30003 to 62209, wherein the 5′ end of the nucleotidesequence provided in any one of SEQ ID NOS: 30003 to 62209 is the 5′ endof the oligonucleotide. In some embodiments, the at least 8 consecutivenucleotides are present within the nucleotide sequence set forth as SEQID NO: 6.

In some embodiments, a single stranded oligonucleotide comprises anucleotide sequence as set forth in any one of SEQ ID NOS: 69 to 62225.In some embodiments, the oligonucleotide is up to 50 nucleotides inlength. In some embodiments, a single stranded oligonucleotide comprisesa fragment of at least 8 nucleotides of a nucleotide sequence as setforth in any one of SEQ ID NOS: 69 to 62225.

In some embodiments, a single stranded oligonucleotide comprises anucleotide sequence as set forth in Table 4. In some embodiments, thesingle stranded oligonucleotide comprises a fragment of at least 8nucleotides of a nucleotide sequence as set forth in Table 4. In someembodiments, a single stranded oligonucleotide consists of a nucleotidesequence as set forth in Table 4.

In some embodiments, the single stranded oligonucleotide does notcomprise three or more consecutive guanosine nucleotides. In someembodiments, the single stranded oligonucleotide does not comprise fouror more consecutive guanosine nucleotides.

In some embodiments, the single stranded oligonucleotide is 8 to 30nucleotides in length. In some embodiments, the single strandedoligonucleotide is up to 50 nucleotides in length. In some embodiments,the single stranded oligonucleotide is 8 to 10 nucleotides in length andall but 1, 2, or 3 of the nucleotides of the complementary sequence ofthe PRC2-associated region are cytosine or guanosine nucleotides.

In some embodiments, the single stranded oligonucleotide iscomplementary with at least 8 consecutive nucleotides of aPRC2-associated region of a BDNF gene, e.g., a PRC2-associated region ofa nucleotide sequence set forth as SEQ ID NO: 1 or 2, in which thenucleotide sequence of the single stranded oligonucleotide comprises oneor more of a nucleotide sequence selected from the group consisting of

(a) (X)Xxxxxx, (X)xXxxxx, (X)xxXxxx, (X)xxxXxx, (X)xxxxXx and (X)xxxxxX,

(b) (X)XXxxxx, (X)XxXxxx, (X)XxxXxx, (X)XxxxXx, (X)XxxxxX, (X)xXXxxx,(X)xXxXxx, (X)xXxxXx, (X)xXxxxX, (X)xxXXxx, (X)xxXxXx, (X)xxXxxX,(X)xxxXXx, (X)xxxXxX and (X)xxxxXX,

(c) (X)XXXxxx, (X)xXXXxx, (X)xxXXXx, (X)xxxXXX, (X)XXxXxx, (X)XXxxXx,(X)XXxxxX, (X)xXXxXx, (X)xXXxxX, (X)xxXXxX, (X)XxXXxx, (X)XxxXXx(X)XxxxXX, (X)xXxXXx, (X)xXxxXX, (X)xxXxXX, (X)xXxXxX and (X)XxXxXx,

(d) (X)xxXXX, (X)xXxXXX, (X)xXXxXX, (X)xXXXxX, (X)xXXXXx, (X)XxxXXXX,(X)XxXxXX, (X)XxXXxX, (X)XxXXx, (X)XXxxXX, (X)XXxXxX, (X)XXxXXx,(X)XXXxxX, (X)XXXxXx, and (X)XXXXxx,

(e) (X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX, (X)XXXXxX and (X)XXXXXx,and

(f) XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxXX, XXXXXxX and XXXXXXx,wherein “X” denotes a nucleotide analogue, (X) denotes an optionalnucleotide analogue, and “x” denotes a DNA or RNA nucleotide unit.

In some embodiments, at least one nucleotide of the oligonucleotide is anucleotide analogue. In some embodiments, the at least one nucleotideanalogue results in an increase in Tm of the oligonucleotide in a rangeof 1 to 5° C. compared with an oligonucleotide that does not have the atleast one nucleotide analogue.

In some embodiments, at least one nucleotide of the oligonucleotidecomprises a 2′ O-methyl. In some embodiments, each nucleotide of theoligonucleotide comprises a 2′ O-methyl. In some embodiments, theoligonucleotide comprises at least one ribonucleotide, at least onedeoxyribonucleotide, or at least one bridged nucleotide. In someembodiments, the bridged nucleotide is a LNA nucleotide, a cEtnucleotide or a ENA modified nucleotide. In some embodiments, eachnucleotide of the oligonucleotide is a LNA nucleotide.

In some embodiments, the nucleotides of the oligonucleotide comprisealternating deoxyribonucleotides and 2′-fluoro-deoxyribonucleotides. Insome embodiments, the nucleotides of the oligonucleotide comprisealternating deoxyribonucleotides and 2′-O-methyl nucleotides. In someembodiments, the nucleotides of the oligonucleotide comprise alternatingdeoxyribonucleotides and ENA nucleotide analogues. In some embodiments,the nucleotides of the oligonucleotide comprise alternatingdeoxyribonucleotides and LNA nucleotides. In some embodiments, the 5′nucleotide of the oligonucleotide is a deoxyribonucleotide. In someembodiments, the nucleotides of the oligonucleotide comprise alternatingLNA nucleotides and 2′-O-methyl nucleotides. In some embodiments, the 5′nucleotide of the oligonucleotide is a LNA nucleotide. In someembodiments, the nucleotides of the oligonucleotide comprisedeoxyribonucleotides flanked by at least one LNA nucleotide on each ofthe 5′ and 3′ ends of the deoxyribonucleotides.

In some embodiments, the single stranded oligonucleotide comprisesmodified internucleotide linkages (e.g., phosphorothioateinternucleotide linkages or other linkages) between at least two, atleast three, at least four, at least five or more nucleotides. In someembodiments, the single stranded oligonucleotide comprises modifiedinternucleotide linkages (e.g., phosphorothioate internucleotidelinkages or other linkages) between between all nucleotides.

In some embodiments, the nucleotide at the 3′ position of theoligonucleotide has a 3′ hydroxyl group. In some embodiments, thenucleotide at the 3′ position of the oligonucleotide has a 3′thiophosphate. In some embodiments, the single stranded oligonucleotidehas a biotin moiety or other moiety conjugated to its 5′ or 3′nucleotide. In some embodiments, the single stranded oligonucleotide hascholesterol, Vitamin A, folate, sigma receptor ligands, aptamers,peptides, such as CPP, hydrophobic molecules, such as lipids, ASGPR ordynamic polyconjugates and variants thereof at its 5′ or 3′ end.

According to some aspects of the invention compositions are providedthat comprise any of the oligonucleotides disclosed herein, and acarrier. In some embodiments, compositions are provided that compriseany of the oligonucleotides in a buffered solution. In some embodiments,the oligonucleotide is conjugated to the carrier. In some embodiments,the carrier is a peptide. In some embodiments, the carrier is a steroid.According to some aspects of the invention pharmaceutical compositionsare provided that comprise any of the oligonucleotides disclosed herein,and a pharmaceutically acceptable carrier.

According to other aspects of the invention, kits are provided thatcomprise a container housing any of the compositions disclosed herein.

According to some aspects of the invention, methods of increasingexpression of BDNF in a cell are provided. In some embodiments, themethods involve delivering any one or more of the single strandedoligonucleotides disclosed herein into the cell. In some embodiments,delivery of the single stranded oligonucleotide into the cell results ina level of expression of BDNF that is greater (e.g., at least 50%greater) than a level of expression of BDNF in a control cell that doesnot comprise the single stranded oligonucleotide.

According to some aspects of the invention, methods of increasing levelsof BDNF in a subject are provided. According to some aspects of theinvention, methods of treating a condition (e.g., neurodegenerationdiseases, such as ALS, AD, HD or PD) associated with decreased levels ofBDNF in a subject are provided. In some embodiments, the methods involveadministering any one or more of the single stranded oligonucleotidesdisclosed herein to the subject.

BRIEF DESCRIPTION OF TABLES

Table 1: Hexamers that are not seed sequences of human miRNAs

Table 2: Oligonucleotide sequences made for testing in the lab. RQ(column 2) and RQ SE (column 3) shows the activity of the oligo relativeto a control well (usually carrier alone) and the standard error or thetriplicate replicates of the experiment. [oligo] is shown in nanomolarfor in vitro experiments and in milligrams per kilogram of body weightfor in vivo experiments. The sequence of each oligonucleotide, includingany modified nucleotides, is shown in Table 4.

Table 3: A listing of oligonucleotide modifications

Table 4: Formatted oligonucleotide sequences made for testing showingnucleotide modifications. The table shows the sequence of the modifiednucleotides, where lnaX represents an LNA nucleotide with 3′phosphorothioate linkage, omeX is a 2′-O-methyl nucleotide, dX is adeoxy nucleotide. An s at the end of a nucleotide code indicates thatthe nucleotide had a 3′ phosphorothioate linkage. The “-Sup” at the endof the sequence marks the fact that the 3′ end lacks either a phosphateor thiophosphate on the 3′ linkage. The Formatted Sequence column showsthe sequence of the oligonucleotide, including modified nucleotides, forthe oligonucleotides tested in Table 2.

Table 5: Cell lines

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Aspects of the invention provided herein relate to the discovery ofpolycomb repressive complex 2 (PRC2)-interacting RNAs. Polycombrepressive complex 2 (PRC2) is a histone methyltransferase and a knownepigenetic regulator involved in silencing of genomic regions throughmethylation of histone H3. Among other functions, PRC2 interacts withlong noncoding RNAs (lncRNAs), such as RepA, Xist, and Tsix, to catalyzetrimethylation of histone H3-lysine27. PRC2 contains four subunits, Eed,Suz12, RbAp48, and Ezh2. Aspects of the invention relate to therecognition that single stranded oligonucleotides that bind toPRC2-associated regions of RNAs (e.g., lncRNAs) that are expressed fromwithin a genomic region that encompasses or that is in functionalproximity to the BDNF gene can induce or enhance expression of BDNF. Insome embodiments, this upregulation is believed to result frominhibition of PRC2 mediated repression of BDNF.

As used herein, the term “PRC2-associated region” refers to a region ofa nucleic acid that comprises or encodes a sequence of nucleotides thatinteract directly or indirectly with a component of PRC2. APRC2-associated region may be present in a RNA (e.g., a long non-codingRNA (lncRNA)) that that interacts with a PRC2. A PRC2-associated regionmay be present in a DNA that encodes an RNA that interacts with PRC2. Insome cases, the PRC2-associated region is equivalently referred to as aPRC2-interacting region.

In some embodiments, a PRC2-associated region is a region of an RNA thatcrosslinks to a component of PRC2 in response to in situ ultravioletirradiation of a cell that expresses the RNA, or a region of genomic DNAthat encodes that RNA region. In some embodiments, a PRC2-associatedregion is a region of an RNA that immunoprecipitates with an antibodythat targets a component of PRC2, or a region of genomic DNA thatencodes that RNA region. In some embodiments, a PRC2-associated regionis a region of an RNA that immunoprecipitates with an antibody thatbinds specifically to SUZ12, EED, EZH2 or RBBP4 (which as noted aboveare components of PRC2), or a region of genomic DNA that encodes thatRNA region.

In some embodiments, a PRC2-associated region is a region of an RNA thatis protected from nucleases (e.g., RNases) in an RNA-immunoprecipitationassay that employs an antibody that targets a component of PRC2, or aregion of genomic DNA that encodes that protected RNA region. In someembodiments, a PRC2-associated region is a region of an RNA that isprotected from nucleases (e.g., RNases) in an RNA-immunoprecipitationassay that employs an antibody that targets SUZ12, EED, EZH2 or RBBP4,or a region of genomic DNA that encodes that protected RNA region.

In some embodiments, a PRC2-associated region is a region of an RNAwithin which occur a relatively high frequency of sequence reads in asequencing reaction of products of an RNA-immunoprecipitation assay thatemploys an antibody that targets a component of PRC2, or a region ofgenomic DNA that encodes that RNA region. In some embodiments, aPRC2-associated region is a region of an RNA within which occur arelatively high frequency of sequence reads in a sequencing reaction ofproducts of an RNA-immunoprecipitation assay that employs an antibodythat binds specifically to SUZ12, EED, EZH2 or RBBP4, or a region ofgenomic DNA that encodes that protected RNA region. In such embodiments,the PRC2-associated region may be referred to as a “peak.”

In some embodiments, a PRC2-associated region comprises a sequence of 40to 60 nucleotides that interact with PRC2 complex. In some embodiments,a PRC2-associated region comprises a sequence of 40 to 60 nucleotidesthat encode an RNA that interacts with PRC2. In some embodiments, aPRC2-associated region comprises a sequence of up to 5 kb in length thatcomprises a sequence (e.g., of 40 to 60 nucleotides) that interacts withPRC2. In some embodiments, a PRC2-associated region comprises a sequenceof up to 5 kb in length within which an RNA is encoded that has asequence (e.g., of 40 to 60 nucleotides) that is known to interact withPRC2. In some embodiments, a PRC2-associated region comprises a sequenceof about 4 kb in length that comprise a sequence (e.g., of 40 to 60nucleotides) that interacts with PRC2. In some embodiments, aPRC2-associated region comprises a sequence of about 4 kb in lengthwithin which an RNA is encoded that includes a sequence (e.g., of 40 to60 nucleotides) that is known to interact with PRC2. In someembodiments, a PRC2-associated region has a sequence as set forth in anyone of SEQ ID NOS: 7 to 68.

In some embodiments, single stranded oligonucleotides are provided thatspecifically bind to, or are complementary to, a PRC2-associated regionin a genomic region that encompasses or that is in proximity to the BDNFgene. In some embodiments, single stranded oligonucleotides are providedthat specifically bind to, or are complementary to, a PRC2-associatedregion that has a sequence as set forth in any one of SEQ ID NOS: 7 to68. In some embodiments, single stranded oligonucleotides are providedthat specifically bind to, or are complementary to, a PRC2-associatedregion that has a sequence as set forth in any one of SEQ ID NOS: 7 to68 combined with up to 2 kb, up to 5 kb, or up to 10 kb of flankingsequences from a corresponding genomic region to which these SEQ IDs map(e.g., in a human genome). In some embodiments, single strandedoligonucleotides have a sequence as set forth in any one of SEQ ID NOS:69 to 62225. In some embodiments, single stranded oligonucleotides havea sequence as set forth in Table 4.

Without being bound by a theory of invention, these oligonucleotides areable to interfere with the binding of and function of PRC2, bypreventing recruitment of PRC2 to a specific chromosomal locus. Forexample, a single administration of single stranded oligonucleotidesdesigned to specifically bind a PRC2-associated region lncRNA can stablydisplace not only the lncRNA, but also the PRC2 that binds to thelncRNA, from binding chromatin. After displacement, the full complementof PRC2 is not recovered for up to 24 hours. Further, lncRNA can recruitPRC2 in a cis fashion, repressing gene expression at or near thespecific chromosomal locus from which the lncRNA was transcribed.

Methods of modulating gene expression are provided, in some embodiments,that may be carried out in vitro, ex vivo, or in vivo. It is understoodthat any reference to uses of compounds throughout the descriptioncontemplates use of the compound in preparation of a pharmaceuticalcomposition or medicament for use in the treatment of condition (e.g.,neurodegeneration diseases, such as ALS, AD, HD or PD) associated withdecreased levels or activity of BDNF. Thus, as one nonlimiting example,this aspect of the invention includes use of such single strandedoligonucleotides in the preparation of a medicament for use in thetreatment of disease, wherein the treatment involves upregulatingexpression of BDNF.

BDNF antisense RNA 1 (BDNF-AS1), also known as BDNF opposite strand(BDNFOS), is a natural antisense transcript to BDNF. BDNF-AS1 hasreverse complementarity with the BDNF coding exon at 224 nucleotides.BDNF-AS1 represses BDNF.

In further aspects of the invention, methods are provided for selectinga candidate oligonucleotide for activating expression of BDNF. Themethods generally involve selecting as a candidate oligonucleotide, asingle stranded oligonucleotide comprising a nucleotide sequence that iscomplementary to a PRC2-associated region (e.g., a nucleotide sequenceas set forth in any one of SEQ ID NOS: 7 to 68). In some embodiments,sets of oligonucleotides may be selected that are enriched (e.g.,compared with a random selection of oligonucleotides) inoligonucleotides that activate expression of BDNF.

Single Stranded Oligonucleotides for Modulating Expression of BDNF

In one aspect of the invention, single stranded oligonucleotidescomplementary to the PRC2-associated regions are provided for modulatingexpression of BDNF in a cell. In some embodiments, expression of BDNF isupregulated or increased. In some embodiments, single strandedoligonucleotides complementary to these PRC2-associated regions inhibitthe interaction of PRC2 with long RNA transcripts such that geneexpression is upregulated or increased. In some embodiments, singlestranded oligonucleotides complementary to these PRC2-associated regionsinhibit the interaction of PRC2 with long RNA transcripts, resulting inreduced methylation of histone H3 and reduced gene inactivation, suchthat gene expression is upregulated or increased. In some embodiments,this interaction may be disrupted or inhibited due to a change in thestructure of the long RNA that prevents or reduces binding to PRC2. Theoligonucleotide may be selected using any of the methods disclosedherein for selecting a candidate oligonucleotide for activatingexpression of BDNF.

The single stranded oligonucleotide may comprise a region ofcomplementarity that is complementary with a PRC2-associated region of anucleotide sequence set forth in any one of SEQ ID NOS: 1 to 6. Theregion of complementarity of the single stranded oligonucleotide may becomplementary with at least 6, e.g., at least 7, at least 8, at least 9,at least 10, at least 15 or more consecutive nucleotides of thePRC2-associated region.

The PRC2-associated region may map to a position in a chromosome between50 kilobases upstream of a 5′-end of the BDNF gene and 50 kilobasesdownstream of a 3′-end of the BDNF gene. The PRC2-associated region maymap to a position in a chromosome between 25 kilobases upstream of a5′-end of the BDNF gene and 25 kilobases downstream of a 3′-end of theBDNF gene. The PRC2-associated region may map to a position in achromosome between 12 kilobases upstream of a 5′-end of the BDNF geneand 12 kilobases downstream of a 3′-end of the BDNF gene. ThePRC2-associated region may map to a position in a chromosome between 5kilobases upstream of a 5′-end of the BDNF gene and 5 kilobasesdownstream of a 3′-end of the BDNF gene.

The genomic position of the selected PRC2-associated region relative tothe BDNF gene may vary. For example, the PRC2-associated region may beupstream of the 5′ end of the BDNF gene. The PRC2-associated region maybe downstream of the 3′ end of the BDNF gene. The PRC2-associated regionmay be within an intron of the BDNF gene. The PRC2-associated region maybe within an exon of the BDNF gene. The PRC2-associated region maytraverse an intron-exon junction, a 5′-UTR-exon junction or a3′-UTR-exon junction of the BDNF gene.

The single stranded oligonucleotide may comprise a sequence having theformula X—Y—Z, in which X is any nucleotide, Y is a nucleotide sequenceof 6 nucleotides in length that is not a human seed sequence of amicroRNA, and Z is a nucleotide sequence of varying length. In someembodiments X is the 5′ nucleotide of the oligonucleotide. In someembodiments, when X is anchored at the 5′ end of the oligonucleotide,the oligonucleotide does not have any nucleotides or nucleotide analogslinked 5′ to X. In some embodiments, other compounds such as peptides orsterols may be linked at the 5′ end in this embodiment as long as theyare not nucleotides or nucleotide analogs. In some embodiments, thesingle stranded oligonucleotide has a sequence 5′X—Y—Z and is 8-50nucleotides in length. Oligonucleotides that have these sequencecharacteristics are predicted to avoid the miRNA pathway. Therefore, insome embodiments, oligonucleotides having these sequence characteristicsare unlikely to have an unintended consequence of functioning in a cellas a miRNA molecule. The Y sequence may be a nucleotide sequence of 6nucleotides in length set forth in Table 1.

The single stranded oligonucleotide may have a sequence that does notcontain guanosine nucleotide stretches (e.g., 3 or more, 4 or more, 5 ormore, 6 or more consecutive guanosine nucleotides). In some embodiments,oligonucleotides having guanosine nucleotide stretches have increasednon-specific binding and/or off-target effects, compared witholigonucleotides that do not have guanosine nucleotide stretches.

The single stranded oligonucleotide may have a sequence that has lessthan a threshold level of sequence identity with every sequence ofnucleotides, of equivalent length, that map to a genomic positionencompassing or in proximity to an off-target gene. For example, anoligonucleotide may be designed to ensure that it does not have asequence that maps to genomic positions encompassing or in proximitywith all known genes (e.g., all known protein coding genes) other thanBDNF. In a similar embodiment, an oligonucleotide may be designed toensure that it does not have a sequence that maps to any other knownPRC2-associated region, particularly PRC2-associated regions that arefunctionally related to any other known gene (e.g., any other knownprotein coding gene). In either case, the oligonucleotide is expected tohave a reduced likelihood of having off-target effects. The thresholdlevel of sequence identity may be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%or 100% sequence identity.

The single stranded oligonucleotide may have a sequence that iscomplementary to a PRC2-associated region that encodes an RNA that formsa secondary structure comprising at least two single stranded loops. Inhas been discovered that, in some embodiments, oligonucleotides that arecomplementary to a PRC2-associated region that encodes an RNA that formsa secondary structure comprising one or more single stranded loops(e.g., at least two single stranded loops) have a greater likelihood ofbeing active (e.g., of being capable of activating or enhancingexpression of a target gene) than a randomly selected oligonucleotide.In some cases, the secondary structure may comprise a double strandedstem between the at least two single stranded loops. Accordingly, theregion of complementarity between the oligonucleotide and thePRC2-associated region may be at a location of the PRC2 associatedregion that encodes at least a portion of at least one of the loops. Insome cases, the region of complementarity between the oligonucleotideand the PRC2-associated region may be at a location of thePRC2-associated region that encodes at least a portion of at least twoof the loops. In some cases, the region of complementarity between theoligonucleotide and the PRC2-associated region may be at a location ofthe PRC2-associated region that encodes at least a portion of the doublestranded stem. In some embodiments, a PRC2-associated region (e.g., ofan lncRNA) is identified (e.g., using RIP-Seq methodology or informationderived therefrom). In some embodiments, the predicted secondarystructure RNA (e.g., lncRNA) containing the PRC2-associated region isdetermined using RNA secondary structure prediction algorithms, e.g.,RNAfold, mfold. In some embodiments, oligonucleotides are designed totarget a region of the RNA that forms a secondary structure comprisingone or more single stranded loop (e.g., at least two single strandedloops) structures which may comprise a double stranded stem between theat least two single stranded loops.

The single stranded oligonucleotide may have a sequence that is hasgreater than 30% G-C content, greater than 40% G-C content, greater than50% G-C content, greater than 60% G-C content, greater than 70% G-Ccontent, or greater than 80% G-C content. The single strandedoligonucleotide may have a sequence that has up to 100% G-C content, upto 95% G-C content, up to 90% G-C content, or up to 80% G-C content. Insome embodiments in which the oligonucleotide is 8 to 10 nucleotides inlength, all but 1, 2, 3, 4, or 5 of the nucleotides of the complementarysequence of the PRC2-associated region are cytosine or guanosinenucleotides. In some embodiments, the sequence of the PRC2-associatedregion to which the single stranded oligonucleotide is complementarycomprises no more than 3 nucleotides selected from adenine and uracil.

The single stranded oligonucleotide may be complementary to a chromosomeof a different species (e.g., a mouse, rat, rabbit, goat, monkey, etc.)at a position that encompasses or that is in proximity to that species'homolog of BDNF. The single stranded oligonucleotide may becomplementary to a human genomic region encompassing or in proximity tothe BDNF gene and also be complementary to a mouse genomic regionencompassing or in proximity to the mouse homolog of BDNF. For example,the single stranded oligonucleotide may be complementary to a sequenceas set forth in SEQ ID NO: 1 or 2, which is a human genomic regionencompassing or in proximity to the BDNF gene, and also be complementaryto a sequence as set forth in SEQ ID NO: 5 or 6, which is a mousegenomic region encompassing or in proximity to the mouse homolog of theBDNF gene. Oligonucleotides having these characteristics may be testedin vivo or in vitro for efficacy in multiple species (e.g., human andmouse). This approach also facilitates development of clinicalcandidates for treating human disease by selecting a species in which anappropriate animal exists for the disease.

In some embodiments, the region of complementarity of the singlestranded oligonucleotide is complementary with at least 8 to 15, 8 to30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 bases, e.g., 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50 consecutive nucleotides of aPRC2-associated region. In some embodiments, the region ofcomplementarity is complementary with at least 8 consecutive nucleotidesof a PRC2-associated region. In some embodiments the sequence of thesingle stranded oligonucleotide is based on an RNA sequence that bindsto PRC2, or a portion thereof, said portion having a length of from 5 to40 contiguous base pairs, or about 8 to 40 bases, or about 5 to 15, orabout 5 to 30, or about 5 to 40 bases, or about 5 to 50 bases.

Complementary, as the term is used in the art, refers to the capacityfor precise pairing between two nucleotides. For example, if anucleotide at a certain position of an oligonucleotide is capable ofhydrogen bonding with a nucleotide at the same position ofPRC2-associated region, then the single stranded nucleotide andPRC2-associated region are considered to be complementary to each otherat that position. The single stranded nucleotide and PRC2-associatedregion are complementary to each other when a sufficient number ofcorresponding positions in each molecule are occupied by nucleotidesthat can hydrogen bond with each other through their bases. Thus,“complementary” is a term which is used to indicate a sufficient degreeof complementarity or precise pairing such that stable and specificbinding occurs between the single stranded nucleotide andPRC2-associated region. For example, if a base at one position of asingle stranded nucleotide is capable of hydrogen bonding with a base atthe corresponding position of a PRC2-associated region, then the basesare considered to be complementary to each other at that position. 100%complementarity is not required.

The single stranded oligonucleotide may be at least 80% complementary to(optionally one of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% complementary to) the consecutive nucleotides of aPRC2-associated region. In some embodiments the single strandedoligonucleotide may contain 1, 2 or 3 base mismatches compared to theportion of the consecutive nucleotides of a PRC2-associated region. Insome embodiments the single stranded oligonucleotide may have up to 3mismatches over 15 bases, or up to 2 mismatches over 10 bases.

It is understood in the art that a complementary nucleotide sequenceneed not be 100% complementary to that of its target to be specificallyhybridizable. In some embodiments, a complementary nucleic acid sequencefor purposes of the present disclosure is specifically hybridizable whenbinding of the sequence to the target molecule (e.g., lncRNA) interfereswith the normal function of the target (e.g., lncRNA) to cause a loss ofactivity (e.g., inhibiting PRC2-associated repression with consequentup-regulation of gene expression) and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the sequence tonon-target sequences under conditions in which avoidance of non-specificbinding is desired, e.g., under physiological conditions in the case ofin vivo assays or therapeutic treatment, and in the case of in vitroassays, under conditions in which the assays are performed undersuitable conditions of stringency.

In some embodiments, the single stranded oligonucleotide is 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50 or more nucleotides in length. In a preferredembodiment, the oligonucleotide is 8 to 30 nucleotides in length.

In some embodiments, the PRC2-associated region occurs on the same DNAstrand as a gene sequence (sense). In some embodiments, thePRC2-associated region occurs on the opposite DNA strand as a genesequence (anti-sense). Oligonucleotides complementary to aPRC2-associated region can bind either sense or anti-sense sequences.Base pairings may include both canonical Watson-Crick base pairing andnon-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteenbase pairing). It is understood that for complementary base pairings,adenosine-type bases (A) are complementary to thymidine-type bases (T)or uracil-type bases (U), that cytosine-type bases (C) are complementaryto guanosine-type bases (G), and that universal bases such as3-nitropyrrole or 5-nitroindole can hybridize to and are consideredcomplementary to any A, C, U, or T. Inosine (I) has also been consideredin the art to be a universal base and is considered complementary to anyA, C, U or T.

In some embodiments, any one or more thymidine (T) nucleotides (ormodified nucleotide thereof) or uridine (U) nucleotides (or a modifiednucleotide thereof) in a sequence provided herein, including a sequenceprovided in the sequence listing, may be replaced with any othernucleotide suitable for base pairing (e.g., via a Watson-Crick basepair) with an adenosine nucleotide. In some embodiments, any one or morethymidine (T) nucleotides (or modified nucleotide thereof) or uridine(U) nucleotides (or a modified nucleotide thereof) in a sequenceprovided herein, including a sequence provided in the sequence listing,may be suitably replaced with a different pyrimidine nucleotide or viceversa. In some embodiments, any one or more thymidine (T) nucleotides(or modified nucleotide thereof) in a sequence provided herein,including a sequence provided in the sequence listing, may be suitablyreplaced with a uridine (U) nucleotide (or a modified nucleotidethereof) or vice versa.

In some embodiments, GC content of the single stranded oligonucleotideis preferably between about 30-60%. Contiguous runs of three or more Gsor Cs may not be preferable in some embodiments. Accordingly, in someembodiments, the oligonucleotide does not comprise a stretch of three ormore guanosine nucleotides.

In some embodiments, the single stranded oligonucleotide specificallybinds to, or is complementary to an RNA that is encoded in a genome(e.g., a human genome) as a single contiguous transcript (e.g., anon-spliced RNA). In some embodiments, the single strandedoligonucleotide specifically binds to, or is complementary to an RNAthat is encoded in a genome (e.g., a human genome), in which thedistance in the genome between the 5′end of the coding region of the RNAand the 3′ end of the coding region of the RNA is less than 1 kb, lessthan 2 kb, less than 3 kb, less than 4 kb, less than 5 kb, less than 7kb, less than 8 kb, less than 9 kb, less than 10 kb, or less than 20 kb.

It is to be understood that any oligonucleotide provided herein can beexcluded. In some embodiments, a single stranded oligonucleotide is notcomplementary to SEQ ID NO: 62226. In some embodiments, a singlestranded oligonucleotide is not complementary to SEQ ID NO: 3.

In some embodiments, a single-stranded oligonucleotide is complementaryto a sequence within nucleotides 1 to 75 or 156 to 4048 of SEQ ID NO:43. In some embodiments, a single-stranded oligonucleotide iscomplementary to a sequence within nucleotides 1 to 1317 or 1370 to 4049of SEQ ID NO: 44. In some embodiments, a single-stranded oligonucleotideis complementary to a sequence within nucleotides 1 to 1766 or 1819 to4083 of SEQ ID NO: 45. In some embodiments, a single-strandedoligonucleotide is complementary to a sequence within nucleotides 1 to1882 or 1935 to 4031 of SEQ ID NO: 46. In some embodiments, asingle-stranded oligonucleotide is complementary to a sequence withinnucleotides 92 to 799 or 859 to 4047 of SEQ ID NO: 52. In someembodiments, a single-stranded oligonucleotide is complementary to asequence within nucleotides 1 to 855, 861 to 863, 1706 to 2546, 2983 to2988, or 3375 to 4045 of SEQ ID NO: 53. In some embodiments, asingle-stranded oligonucleotide is complementary to a sequence withinnucleotides 1 to 856, 920 to 2439, or 2487 to 4050 of SEQ ID NO: 59. Insome embodiments, a single-stranded oligonucleotide is complementary toa sequence within nucleotides 1 to 752, 816 to 2335, or 2383 to 4043 ofSEQ ID NO: 60.

In some embodiments, it has been found that single strandedoligonucleotides disclosed herein may increase expression of mRNAcorresponding to the gene by at least about 50% (i.e. 150% of normal or1.5 fold), or by about 2 fold to about 5 fold. In some embodiments,expression may be increased by at least about 15 fold, 20 fold, 30 fold,40 fold, 50 fold or 100 fold, or any range between any of the foregoingnumbers. It has also been found that increased mRNA expression has beenshown to correlate to increased protein expression.

In some or any of the embodiments of the oligonucleotides describedherein, or processes for designing or synthesizing them, theoligonucleotides will upregulate gene expression and may specificallybind or specifically hybridize or be complementary to the PRC2 bindingRNA that is transcribed from the same strand as a protein codingreference gene. The oligonucleotide may bind to a region of the PRC2binding RNA that originates within or overlaps an intron, exon, intronexon junction, 5′ UTR, 3′ UTR, a translation initiation region, or atranslation termination region of a protein coding sense strand of areference gene (refGene).

In some or any of the embodiments of oligonucleotides described herein,or processes for designing or synthesizing them, the oligonucleotideswill upregulate gene expression and may specifically bind orspecifically hybridize or be complementary to a PRC2 binding RNA thattranscribed from the opposite strand (the antisense strand) of a proteincoding reference gene. The oligonucleotide may bind to a region of thePRC2 binding RNA that originates within or overlaps an intron, exon,intron exon junction, 5′ UTR, 3′ UTR, a translation initiation region,or a translation termination region of a protein coding antisense strandof a reference gene

The oligonucleotides described herein may be modified, e.g., comprise amodified sugar moiety, a modified internucleoside linkage, a modifiednucleotide and/or combinations thereof. In addition, theoligonucleotides can exhibit one or more of the following properties: donot induce substantial cleavage or degradation of the target RNA; do notcause substantially complete cleavage or degradation of the target RNA;do not activate the RNAse H pathway; do not activate RISC; do notrecruit any Argonaute family protein; are not cleaved by Dicer; do notmediate alternative splicing; are not immune stimulatory; are nucleaseresistant; have improved cell uptake compared to unmodifiedoligonucleotides; are not toxic to cells or mammals; may have improvedendosomal exit; do interfere with interaction of lncRNA with PRC2,preferably the Ezh2 subunit but optionally the Suz12, Eed, RbAp46/48subunits or accessory factors such as Jarid2; do decrease histone H3lysine27 methylation and/or do upregulate gene expression.

Oligonucleotides that are designed to interact with RNA to modulate geneexpression are a distinct subset of base sequences from those that aredesigned to bind a DNA target (e.g., are complementary to the underlyinggenomic DNA sequence from which the RNA is transcribed).

Any of the oligonucleotides disclosed herein may be linked to one ormore other oligonucleotides disclosed herein by a linker, e.g., acleavable linker.

Method for Selecting Candidate Oligonucleotides for ActivatingExpression of BDNF

Methods are provided herein for selecting a candidate oligonucleotidefor activating or enhancing expression of BDNF. The target selectionmethods may generally involve steps for selecting single strandedoligonucleotides having any of the structural and functionalcharacteristics disclosed herein. Typically, the methods involve one ormore steps aimed at identifying oligonucleotides that target aPRC2-associated region that is functionally related to BDNF, for examplea PRC2-associated region of a lncRNA that regulates expression of BDNFby facilitating (e.g., in a cis-regulatory manner) the recruitment ofPRC2 to the BDNF gene. Such oligonucleotides are expected to becandidates for activating expression of BDNF because of their ability tohybridize with the PRC2-associated region of a nucleic acid (e.g., alncRNA). In some embodiments, this hybridization event is understood todisrupt interaction of PRC2 with the nucleic acid (e.g., a lncRNA) andas a result disrupt recruitment of PRC2 and its associated co-repressors(e.g., chromatin remodeling factors) to the BDNF gene locus.

Methods of selecting a candidate oligonucleotide may involve selecting aPRC2-associated region (e.g., a nucleotide sequence as set forth in anyone of SEQ ID NOS: 7 to 68) that maps to a chromosomal positionencompassing or in proximity to the BDNF gene (e.g., a chromosomalposition having a sequence as set forth in any one of SEQ ID NOS: 1 to6). The PRC2-associated region may map to the strand of the chromosomecomprising the sense strand of the BDNF gene, in which case thecandidate oligonucleotide is complementary to the sense strand of theBDNF gene (i.e., is antisense to the BDNF gene). Alternatively, thePRC2-associated region may map to the strand of the first chromosomecomprising the antisense strand of the BDNF gene, in which case theoligonucleotide is complementary to the antisense strand (the templatestrand) of the BDNF gene (i.e., is sense to the BDNF gene).

Methods for selecting a set of candidate oligonucleotides that isenriched in oligonucleotides that activate expression of BDNF mayinvolve selecting one or more PRC2-associated regions that map to achromosomal position that encompasses or that is in proximity to theBDNF gene and selecting a set of oligonucleotides, in which eacholigonucleotide in the set comprises a nucleotide sequence that iscomplementary with the one or more PRC2-associated regions. As usedherein, the phrase, “a set of oligonucleotides that is enriched inoligonucleotides that activate expression of” refers to a set ofoligonucleotides that has a greater number of oligonucleotides thatactivate expression of a target gene (e.g., BDNF) compared with a randomselection of oligonucleotides of the same physicochemical properties(e.g., the same GC content, T_(m), length etc.) as the enriched set.

Where the design and/or synthesis of a single stranded oligonucleotideinvolves design and/or synthesis of a sequence that is complementary toa nucleic acid or PRC2-associated region described by such sequenceinformation, the skilled person is readily able to determine thecomplementary sequence, e.g., through understanding of Watson Crick basepairing rules which form part of the common general knowledge in thefield.

In some embodiments design and/or synthesis of a single strandedoligonucleotide involves manufacture of an oligonucleotide from startingmaterials by techniques known to those of skill in the art, where thesynthesis may be based on a sequence of a PRC2-associated region, orportion thereof.

Methods of design and/or synthesis of a single stranded oligonucleotidemay involve one or more of the steps of:

Identifying and/or selecting PRC2-associated region;

Designing a nucleic acid sequence having a desired degree of sequenceidentity or complementarity to a PRC2-associated region or a portionthereof;

Synthesizing a single stranded oligonucleotide to the designed sequence;

Purifying the synthesized single stranded oligonucleotide; and

Optionally mixing the synthesized single stranded oligonucleotide withat least one pharmaceutically acceptable diluent, carrier or excipientto form a pharmaceutical composition or medicament.

Single stranded oligonucleotides so designed and/or synthesized may beuseful in method of modulating gene expression as described herein.

Preferably, single stranded oligonucleotides of the invention aresynthesized chemically. Oligonucleotides used to practice this inventioncan be synthesized in vitro by well-known chemical synthesis techniques.

Oligonucleotides of the invention can be stabilized against nucleolyticdegradation such as by the incorporation of a modification, e.g., anucleotide modification. For example, nucleic acid sequences of theinvention include a phosphorothioate at least the first, second, orthird internucleotide linkage at the 5′ or 3′ end of the nucleotidesequence. As another example, the nucleic acid sequence can include a2′-modified nucleotide, e.g., a 2′-deoxy, 2′-deoxy-2′-fluoro,2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP),2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), or2′-O—N-methylacetamido (2′-O-NMA). As another example, the nucleic acidsequence can include at least one 2′-O-methyl-modified nucleotide, andin some embodiments, all of the nucleotides include a 2′-O-methylmodification. In some embodiments, the nucleic acids are “locked,” i.e.,comprise nucleic acid analogues in which the ribose ring is “locked” bya methylene bridge connecting the 2′-O atom and the 4′-C atom.

It is understood that any of the modified chemistries or formats ofsingle stranded oligonucleotides described herein can be combined witheach other, and that one, two, three, four, five, or more differenttypes of modifications can be included within the same molecule.

In some embodiments, the method may further comprise the steps ofamplifying the synthesized single stranded oligonucleotide, and/orpurifying the single stranded oligonucleotide (or amplified singlestranded oligonucleotide), and/or sequencing the single strandedoligonucleotide so obtained.

As such, the process of preparing a single stranded oligonucleotide maybe a process that is for use in the manufacture of a pharmaceuticalcomposition or medicament for use in the treatment of disease,optionally wherein the treatment involves modulating expression of agene associated with a PRC2-associated region.

In the methods described above a PRC2-associated region may be, or havebeen, identified, or obtained, by a method that involves identifying RNAthat binds to PRC2.

Such methods may involve the following steps: providing a samplecontaining nuclear ribonucleic acids, contacting the sample with anagent that binds specifically to PRC2 or a subunit thereof, allowingcomplexes to form between the agent and protein in the sample,partitioning the complexes, synthesizing nucleic acid that iscomplementary to nucleic acid present in the complexes.

Where the single stranded oligonucleotide is based on a PRC2-associatedregion, or a portion of such a sequence, it may be based on informationabout that sequence, e.g., sequence information available in written orelectronic form, which may include sequence information contained inpublicly available scientific publications or sequence databases.

Nucleotide Analogues

In some embodiments, the oligonucleotide may comprise at least oneribonucleotide, at least one deoxyribonucleotide, and/or at least onebridged nucleotide. In some embodiments, the oligonucleotide maycomprise a bridged nucleotide, such as a locked nucleic acid (LNA)nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene bridgednucleic acid (ENA) nucleotide. Examples of such nucleotides aredisclosed herein and known in the art. In some embodiments, theoligonucleotide comprises a nucleotide analog disclosed in one of thefollowing United States patent or Patent Application Publications: U.S.Pat. No. 7,399,845, U.S. Pat. No. 7,741,457, U.S. Pat. No. 8,022,193,U.S. Pat. No. 7,569,686, U.S. Pat. No. 7,335,765, U.S. Pat. No.7,314,923, U.S. Pat. No. 7,335,765, and U.S. Pat. No. 7,816,333, US20110009471, the entire contents of each of which are incorporatedherein by reference for all purposes. The oligonucleotide may have oneor more 2′ O-methyl nucleotides. The oligonucleotide may consistentirely of 2′ O-methyl nucleotides.

Often the single stranded oligonucleotide has one or more nucleotideanalogues. For example, the single stranded oligonucleotide may have atleast one nucleotide analogue that results in an increase in T_(m) ofthe oligonucleotide in a range of 1° C., 2° C., 3° C., 4° C., or 5° C.compared with an oligonucleotide that does not have the at least onenucleotide analogue. The single stranded oligonucleotide may have aplurality of nucleotide analogues that results in a total increase inT_(m) of the oligonucleotide in a range of 2° C., 3° C., 4° C., 5° C.,6° C., 7° C., 8° C., 9° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35°C., 40° C., 45° C. or more compared with an oligonucleotide that doesnot have the nucleotide analogue.

The oligonucleotide may be of up to 50 nucleotides in length in which 2to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2to 30, 2 to 40, 2 to 45, or more nucleotides of the oligonucleotide arenucleotide analogues. The oligonucleotide may be of 8 to 30 nucleotidesin length in which 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19,2 to 20, 2 to 25, 2 to 30 nucleotides of the oligonucleotide arenucleotide analogues.

The oligonucleotide may be of 8 to 15 nucleotides in length in which 2to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12,2 to 13, 2 to 14 nucleotides of the oligonucleotide are nucleotideanalogues. Optionally, the oligonucleotides may have every nucleotideexcept 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides modified.

The oligonucleotide may consist entirely of bridged nucleotides (e.g.,LNA nucleotides, cEt nucleotides, ENA nucleotides). The oligonucleotidemay comprise alternating deoxyribonucleotides and2′-fluoro-deoxyribonucleotides. The oligonucleotide may comprisealternating deoxyribonucleotides and 2′-O-methyl nucleotides. Theoligonucleotide may comprise alternating deoxyribonucleotides and ENAnucleotide analogues. The oligonucleotide may comprise alternatingdeoxyribonucleotides and LNA nucleotides. The oligonucleotide maycomprise alternating LNA nucleotides and 2′-O-methyl nucleotides. Theoligonucleotide may have a 5′ nucleotide that is a bridged nucleotide(e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide). Theoligonucleotide may have a 5′ nucleotide that is a deoxyribonucleotide.

The oligonucleotide may comprise deoxyribonucleotides flanked by atleast one bridged nucleotide (e.g., a LNA nucleotide, cEt nucleotide,ENA nucleotide) on each of the 5′ and 3′ ends of thedeoxyribonucleotides. The oligonucleotide may comprisedeoxyribonucleotides flanked by 1, 2, 3, 4, 5, 6, 7, 8 or more bridgednucleotides (e.g., LNA nucleotides, cEt nucleotides, ENA nucleotides) oneach of the 5′ and 3′ ends of the deoxyribonucleotides. The 3′ positionof the oligonucleotide may have a 3′ hydroxyl group. The 3′ position ofthe oligonucleotide may have a 3′ thiophosphate.

The oligonucleotide may be conjugated with a label. For example, theoligonucleotide may be conjugated with a biotin moiety, cholesterol,Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such asCPP, hydrophobic molecules, such as lipids, ASGPR or dynamicpolyconjugates and variants thereof at its 5′ or 3′ end.

Preferably the single stranded oligonucleotide comprises one or moremodifications comprising: a modified sugar moiety, and/or a modifiedinternucleoside linkage, and/or a modified nucleotide and/orcombinations thereof. It is not necessary for all positions in a givenoligonucleotide to be uniformly modified, and in fact more than one ofthe modifications described herein may be incorporated in a singleoligonucleotide or even at within a single nucleoside within anoligonucleotide.

In some embodiments, the single stranded oligonucleotides are chimericoligonucleotides that contain two or more chemically distinct regions,each made up of at least one nucleotide. These oligonucleotidestypically contain at least one region of modified nucleotides thatconfers one or more beneficial properties (such as, for example,increased nuclease resistance, increased uptake into cells, increasedbinding affinity for the target) and a region that is a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric singlestranded oligonucleotides of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures comprise, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,each of which is herein incorporated by reference.

In some embodiments, the single stranded oligonucleotide comprises atleast one nucleotide modified at the 2′ position of the sugar, mostpreferably a 2′-O-alkyl, 2′-O-alkyl-O-alkyl or 2′-fluoro-modifiednucleotide. In other preferred embodiments, RNA modifications include2′-fluoro, 2′-amino and 2′ O-methyl modifications on the ribose ofpyrimidines, abasic residues or an inverted base at the 3′ end of theRNA. Such modifications are routinely incorporated into oligonucleotidesand these oligonucleotides have been shown to have a higher Tm (i.e.,higher target binding affinity) than 2′-deoxyoligonucleotides against agiven target.

A number of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligodeoxynucleotide; thesemodified oligos survive intact for a longer time than unmodifiedoligonucleotides. Specific examples of modified oligonucleotides includethose comprising modified backbones, for example, phosphorothioates,phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. Most preferred are oligonucleotides withphosphorothioate backbones and those with heteroatom backbones,particularly CH₂—NH—O—CH₂, CH,˜N(CH₃)˜O˜CH₂ (known as amethylene(methylimino) or MMI backbone, CH₂—O—N(CH₃)—CH₂,CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH,); amide backbones(see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholinobackbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506);peptide nucleic acid (PNA) backbone (wherein the phosphodiester backboneof the oligonucleotide is replaced with a polyamide backbone, thenucleotides being bound directly or indirectly to the aza nitrogen atomsof the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).Phosphorus-containing linkages include, but are not limited to,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates comprising 3′alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates comprising 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′; see U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563, 253; 5,571,799;5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Dwaine A. Braaschand David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis,volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214;Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc.Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506,issued Jul. 23, 1991. In some embodiments, the morpholino-basedoligomeric compound is a phosphorodiamidate morpholino oligomer (PMO)(e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001;and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures ofwhich are incorporated herein by reference in their entireties).

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc., 2000, 122, 8595-8602.

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439, each of which is herein incorporated by reference.

Modified oligonucleotides are also known that include oligonucleotidesthat are based on or constructed from arabinonucleotide or modifiedarabinonucleotide residues. Arabinonucleosides are stereoisomers ofribonucleosides, differing only in the configuration at the 2′-positionof the sugar ring. In some embodiments, a 2′-arabino modification is2′-F arabino. In some embodiments, the modified oligonucleotide is2′-fluoro-D-arabinonucleic acid (FANA) (as described in, for example,Lon et al., Biochem., 41:3457-3467, 2002 and Min et al., Bioorg. Med.Chem. Lett., 12:2651-2654, 2002; the disclosures of which areincorporated herein by reference in their entireties). Similarmodifications can also be made at other positions on the sugar,particularly the 3′ position of the sugar on a 3′ terminal nucleoside orin 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminalnucleotide.

PCT Publication No. WO 99/67378 discloses arabinonucleic acids (ANA)oligomers and their analogues for improved sequence specific inhibitionof gene expression via association to complementary messenger RNA.

Other preferred modifications include ethylene-bridged nucleic acids(ENAs) (e.g., International Patent Publication No. WO 2005/042777,Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al.,Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther.,8:144-149, 2006 and Horie et al., Nucleic Acids Symp. Ser (Oxf),49:171-172, 2005; the disclosures of which are incorporated herein byreference in their entireties). Preferred ENAs include, but are notlimited to, 2′-0,4′-C-ethylene-bridged nucleic acids.

Examples of LNAs are described in WO/2008/043753 and include compoundsof the following general formula.

where X and Y are independently selected among the groups —O—,

—S—, —N(H)—, N(R)—, —CH₂— or —CH— (if part of a double bond),

—CH₂—O—, —CH₂—S—, —CH₂—N(H)—, —CH₂—N(R)—, —CH₂—CH₂— or —CH₂—CH— (if partof a double bond),

—CH═CH—, where R is selected from hydrogen and C₁₋₄-alkyl; Z and Z* areindependently selected among an internucleoside linkage, a terminalgroup or a protecting group; B constitutes a natural or non-naturalnucleotide base moiety; and the asymmetric groups may be found in eitherorientation.

Preferably, the LNA used in the oligonucleotides described hereincomprises at least one LNA unit according any of the formulas

wherein Y is —O—, —S—, —NH—, or N(R^(H)); Z and Z* are independentlyselected among an internucleoside linkage, a terminal group or aprotecting group; B constitutes a natural or non-natural nucleotide basemoiety, and RH is selected from hydrogen and C₁₋₄-alkyl.

In some embodiments, the Locked Nucleic Acid (LNA) used in theoligonucleotides described herein comprises at least one Locked NucleicAcid (LNA) unit according any of the formulas shown in Scheme 2 ofPCT/DK2006/000512.

In some embodiments, the LNA used in the oligomer of the inventioncomprises internucleoside linkages selected from —O—P(O)₂—O—,—O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—,—O—P(O)₂—S—, —O—P(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—, O—PO(OCH₃)—O—,—O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—,where R^(H) is selected from hydrogen and C₁₋₄-alkyl.

Specifically preferred LNA units are shown in scheme 2:

The term “thio-LNA” comprises a locked nucleotide in which at least oneof X or Y in the general formula above is selected from S or —CH₂—S—.Thio-LNA can be in both beta-D and alpha-L-configuration.

The term “amino-LNA” comprises a locked nucleotide in which at least oneof X or Y in the general formula above is selected from —N(H)—, N(R)—,CH₂—N(H)—, and —CH₂—N(R)— where R is selected from hydrogen andC₁₋₄-alkyl. Amino-LNA can be in both beta-D and alpha-L-configuration.

The term “oxy-LNA” comprises a locked nucleotide in which at least oneof X or Y in the general formula above represents —O— or —CH₂—O—.Oxy-LNA can be in both beta-D and alpha-L-configuration.

The term “ena-LNA” comprises a locked nucleotide in which Y in thegeneral formula above is —CH₂—O— (where the oxygen atom of —CH₂—O— isattached to the 2′-position relative to the base B).

LNAs are described in additional detail herein.

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃ OCH₃,OCH₃ O(CH₂)n CH₃, O(CH₂)n NH₂ or O(CH₂)n CH₃ where n is from 1 to about10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O-, S-, or N-alkyl; O-, S-,or N-alkenyl; SOCH₃; SO₂ CH₃; ONO₂; NO₂; N₃; NH2; heterocycloalkyl;heterocycloalkaryl; amino alkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. A preferredmodification includes 2′-methoxyethoxy[2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486).Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-propoxy(2′-OCH₂ CH₂CH₃) and 2′-fluoro (2′-F). Similar modifications may also bemade at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide and the 5′ positionof 5′ terminal nucleotide. Oligonucleotides may also have sugar mimeticssuch as cyclobutyls in place of the pentofuranosyl group.

Single stranded oligonucleotides can also include, additionally oralternatively, nucleobase (often referred to in the art simply as“base”) modifications or substitutions. As used herein, “unmodified” or“natural” nucleobases include adenine (A), guanine (G), thymine (T),cytosine (C) and uracil (U). Modified nucleobases include nucleobasesfound only infrequently or transiently in natural nucleic acids, e.g.,hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine andoften referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC),glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, aswell as synthetic nucleobases, e.g., 2-aminoadenine,2-(methylamino)adenine, 2-(imidazolylalkyl)adenine,2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines,2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil,5-propynyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, 6-aminopurine, 2-aminopurine,2-chloro-6-aminopurine and 2,6-diaminopurine or other diaminopurines.See, e.g., Kornberg, “DNA Replication,” W. H. Freeman & Co., SanFrancisco, 1980, pp 75-77; and Gebeyehu, G., et al. Nucl. Acids Res.,15:4513 (1987)). A “universal” base known in the art, e.g., inosine, canalso be included. 5-Me-C substitutions have been shown to increasenucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, in Crooke, andLebleu, eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and may be used as base substitutions.

It is not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the modificationsdescribed herein may be incorporated in a single oligonucleotide or evenat within a single nucleoside within an oligonucleotide.

In some embodiments, both a sugar and an internucleoside linkage, i.e.,the backbone, of the nucleotide units are replaced with novel groups.The base units are maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, anoligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide isreplaced with an amide containing backbone, for example, anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al, Science, 1991, 254, 1497-1500.

Single stranded oligonucleotides can also include one or more nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasescomprise the purine bases adenine (A) and guanine (G), and thepyrimidine bases thymine (T), cytosine (C) and uracil (U). Modifiednucleobases comprise other synthetic and natural nucleobases such as5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouraciland cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylquanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Further, nucleobases comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in “The Concise Encyclopedia of PolymerScience And Engineering”, pages 858-859, Kroschwitz, ed. John Wiley &Sons, 1990; those disclosed by Englisch et al., Angewandle Chemie,International Edition, 1991, 30, page 613, and those disclosed bySanghvi, Chapter 15, Antisense Research and Applications,” pages289-302, Crooke, and Lebleu, eds., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2<0>C (Sanghvi, etal., eds, “Antisense Research and Applications,” CRC Press, Boca Raton,1993, pp. 276-278) and are presently preferred base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications. Modified nucleobases are described in U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617;5,750,692, and 5,681,941, each of which is herein incorporated byreference.

In some embodiments, the single stranded oligonucleotides are chemicallylinked to one or more moieties or conjugates that enhance the activity,cellular distribution, or cellular uptake of the oligonucleotide. Forexample, one or more single stranded oligonucleotides, of the same ordifferent types, can be conjugated to each other; or single strandedoligonucleotides can be conjugated to targeting moieties with enhancedspecificity for a cell type or tissue type. Such moieties include, butare not limited to, lipid moieties such as a cholesterol moiety(Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556),cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4,1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al,Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-toxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996,277, 923-937). See also U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250;5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463;5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142;5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and5,688,941, each of which is herein incorporated by reference.

These moieties or conjugates can include conjugate groups covalentlybound to functional groups such as primary or secondary hydroxyl groups.Conjugate groups of the invention include intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, polyethers,groups that enhance the pharmacodynamic properties of oligomers, andgroups that enhance the pharmacokinetic properties of oligomers. Typicalconjugate groups include cholesterols, lipids, phospholipids, biotin,phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties, in the context of this invention, includegroups that improve uptake, enhance resistance to degradation, and/orstrengthen sequence-specific hybridization with the target nucleic acid.Groups that enhance the pharmacokinetic properties, in the context ofthis invention, include groups that improve uptake, distribution,metabolism or excretion of the compounds of the present invention.Representative conjugate groups are disclosed in International PatentApplication No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No.6,287,860, which are incorporated herein by reference. Conjugatemoieties include, but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or adamantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941.

In some embodiments, single stranded oligonucleotide modificationinclude modification of the 5′ or 3′ end of the oligonucleotide. In someembodiments, the 3′ end of the oligonucleotide comprises a hydroxylgroup or a thiophosphate. It should be appreciated that additionalmolecules (e.g. a biotin moiety or a fluorophor) can be conjugated tothe 5′ or 3′ end of the single stranded oligonucleotide. In someembodiments, the single stranded oligonucleotide comprises a biotinmoiety conjugated to the 5′ nucleotide.

In some embodiments, the single stranded oligonucleotide compriseslocked nucleic acids (LNA), ENA modified nucleotides, 2′-O-methylnucleotides, or 2′-fluoro-deoxyribonucleotides. In some embodiments, thesingle stranded oligonucleotide comprises alternatingdeoxyribonucleotides and 2′-fluoro-deoxyribonucleotides. In someembodiments, the single stranded oligonucleotide comprises alternatingdeoxyribonucleotides and 2′-O-methyl nucleotides. In some embodiments,the single stranded oligonucleotide comprises alternatingdeoxyribonucleotides and ENA modified nucleotides. In some embodiments,the single stranded oligonucleotide comprises alternatingdeoxyribonucleotides and locked nucleic acid nucleotides. In someembodiments, the single stranded oligonucleotide comprises alternatinglocked nucleic acid nucleotides and 2′-O-methyl nucleotides.

In some embodiments, the 5′ nucleotide of the oligonucleotide is adeoxyribonucleotide. In some embodiments, the 5′ nucleotide of theoligonucleotide is a locked nucleic acid nucleotide. In someembodiments, the nucleotides of the oligonucleotide comprisedeoxyribonucleotides flanked by at least one locked nucleic acidnucleotide on each of the 5′ and 3′ ends of the deoxyribonucleotides. Insome embodiments, the nucleotide at the 3′ position of theoligonucleotide has a 3′ hydroxyl group or a 3′ thiophosphate.

In some embodiments, the single stranded oligonucleotide comprisesphosphorothioate internucleotide linkages. In some embodiments, thesingle stranded oligonucleotide comprises phosphorothioateinternucleotide linkages between at least two nucleotides. In someembodiments, the single stranded oligonucleotide comprisesphosphorothioate internucleotide linkages between all nucleotides.

It should be appreciated that the single stranded oligonucleotide canhave any combination of modifications as described herein.

The oligonucleotide may comprise a nucleotide sequence having one ormore of the following modification patterns.

(a) (X)Xxxxxx, (X)xXxxxx, (X)xxXxxx, (X)xxxXxx, (X)xxxxXx and (X)xxxxxX,

(b) (X)XXxxxx, (X)XxXxxx, (X)XxxXxx, (X)XxxxXx, (X)XxxxxX, (X)xXXxxx,(X)xXxXxx, (X)xXxxXx, (X)xXxxxX, (X)xxXXxx, (X)xxXxXx, (X)xxXxxX,(X)xxxXXx, (X)xxxXxX and (X)xxxxXX,

(c) (X)XXXxxx, (X)xXXXxx, (X)xxXXXx, (X)xxxXXX, (X)XXxXxx, (X)XXxxXx,(X)XXxxxX, (X)xXXxXx, (X)xXXxxX, (X)xxXXxX, (X)XxXXxx, (X)XxxXXx(X)XxxxXX, (X)xXxXXx, (X)xXxxXX, (X)xxXxXX, (X)xXxXxX and (X)XxXxXx,

(d) (X)xxXXX, (X)xXxXXX, (X)xXXxXX, (X)xXXXxX, (X)xXXXXx, (X)XxxXXXX,(X)XxXxXX, (X)XxXXxX, (X)XxXXx, (X)XXxxXX, (X)XXxXxX,

(X)XXxXXx, (X)XXXxxX, (X)XXXxXx, and (X)XXXXxx,

(e) (X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX, (X)XXXXxX and (X)XXXXXx,and

(f) XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxXX, XXXXXxX and XXXXXXx, inwhich “X” denotes a nucleotide analogue, (X) denotes an optionalnucleotide analogue, and “x” denotes a DNA or RNA nucleotide unit. Eachof the above listed patterns may appear one or more times within anoligonucleotide, alone or in combination with any of the other disclosedmodification patterns.

Methods for Modulating Gene Expression

In one aspect, the invention relates to methods for modulating geneexpression in a cell (e.g., a cell for which BDNF levels are reduced)for research purposes (e.g., to study the function of the gene in thecell). In another aspect, the invention relates to methods formodulating gene expression in a cell (e.g., a cell for which BDNF levelsare reduced) for gene or epigenetic therapy. The cells can be in vitro,ex vivo, or in vivo (e.g., in a subject who has a disease resulting fromreduced expression or activity of BDNF. In some embodiments, methods formodulating gene expression in a cell comprise delivering a singlestranded oligonucleotide as described herein. In some embodiments,delivery of the single stranded oligonucleotide to the cell results in alevel of expression of gene that is at least 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 100%, 200% or more greater than a level ofexpression of gene in a control cell to which the single strandedoligonucleotide has not been delivered. In certain embodiments, deliveryof the single stranded oligonucleotide to the cell results in a level ofexpression of gene that is at least 50% greater than a level ofexpression of gene in a control cell to which the single strandedoligonucleotide has not been delivered.

In another aspect of the invention, methods comprise administering to asubject (e.g. a human) a composition comprising a single strandedoligonucleotide as described herein to increase protein levels in thesubject. In some embodiments, the increase in protein levels is at least5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or more,higher than the amount of a protein in the subject before administering.

As another example, to increase expression of BDNF in a cell, themethods include introducing into the cell a single strandedoligonucleotide that is sufficiently complementary to a PRC2-associatedregion (e.g., of a long non-coding RNA) that maps to a genomic positionencompassing or in proximity to the BDNF gene.

In another aspect of the invention provides methods of treating acondition (e.g., neurodegeneration diseases, such as ALS, AD, HD or PD)associated with decreased levels of expression of BDNF in a subject, themethod comprising administering a single stranded oligonucleotide asdescribed herein.

A subject can include a non-human mammal, e.g. mouse, rat, guinea pig,rabbit, cat, dog, goat, cow, or horse. In preferred embodiments, asubject is a human. Single stranded oligonucleotides have been employedas therapeutic moieties in the treatment of disease states in animals,including humans. Single stranded oligonucleotides can be usefultherapeutic modalities that can be configured to be useful in treatmentregimes for the treatment of cells, tissues and animals, especiallyhumans.

For therapeutics, an animal, preferably a human, suspected of havingneurodegeneration diseases, such as ALS, AD, HD or PD is treated byadministering single stranded oligonucleotide in accordance with thisinvention. For example, in one non-limiting embodiment, the methodscomprise the step of administering to the animal in need of treatment, atherapeutically effective amount of a single stranded oligonucleotide asdescribed herein.

Formulation, Delivery, And Dosing

The oligonucleotides described herein can be formulated foradministration to a subject for treating a condition (e.g.,neurodegeneration diseases, such as ALS, AD, HD or PD) associated withdecreased levels of BDNF. It should be understood that the formulations,compositions and methods can be practiced with any of theoligonucleotides disclosed herein.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient (e.g., an oligonucleotide or compound of theinvention) which can be combined with a carrier material to produce asingle dosage form will vary depending upon the host being treated, theparticular mode of administration, e.g., intradermal or inhalation. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the compound which produces a therapeutic effect, e.g. tumorregression.

Pharmaceutical formulations of this invention can be prepared accordingto any method known to the art for the manufacture of pharmaceuticals.Such formulations can contain sweetening agents, flavoring agents,coloring agents and preserving agents. A formulation can be admixturedwith nontoxic pharmaceutically acceptable excipients which are suitablefor manufacture. Formulations may comprise one or more diluents,emulsifiers, preservatives, buffers, excipients, etc. and may beprovided in such forms as liquids, powders, emulsions, lyophilizedpowders, sprays, creams, lotions, controlled release formulations,tablets, pills, gels, on patches, in implants, etc.

A formulated single stranded oligonucleotide composition can assume avariety of states. In some examples, the composition is at leastpartially crystalline, uniformly crystalline, and/or anhydrous (e.g.,less than 80, 50, 30, 20, or 10% water). In another example, the singlestranded oligonucleotide is in an aqueous phase, e.g., in a solutionthat includes water. The aqueous phase or the crystalline compositionscan, e.g., be incorporated into a delivery vehicle, e.g., a liposome(particularly for the aqueous phase) or a particle (e.g., amicroparticle as can be appropriate for a crystalline composition).Generally, the single stranded oligonucleotide composition is formulatedin a manner that is compatible with the intended method ofadministration.

In some embodiments, the composition is prepared by at least one of thefollowing methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

A single stranded oligonucleotide preparation can be formulated oradministered (together or separately) in combination with another agent,e.g., another therapeutic agent or an agent that stabilizes a singlestranded oligonucleotide, e.g., a protein that complexes with singlestranded oligonucleotide. Still other agents include chelators, e.g.,EDTA (e.g., to remove divalent cations such as Mg²⁺), salts, RNAseinhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin)and so forth.

In one embodiment, the single stranded oligonucleotide preparationincludes another single stranded oligonucleotide, e.g., a second singlestranded oligonucleotide that modulates expression of a second gene or asecond single stranded oligonucleotide that modulates expression of thefirst gene. Still other preparation can include at least 3, 5, ten,twenty, fifty, or a hundred or more different single strandedoligonucleotide species. Such single stranded oligonucleotides canmediated gene expression with respect to a similar number of differentgenes. In one embodiment, the single stranded oligonucleotidepreparation includes at least a second therapeutic agent (e.g., an agentother than an oligonucleotide).

Route of Delivery

A composition that includes a single stranded oligonucleotide can bedelivered to a subject by a variety of routes. Exemplary routes include:intravenous, intradermal, topical, rectal, parenteral, anal,intravaginal, intranasal, pulmonary, ocular. The term “therapeuticallyeffective amount” is the amount of oligonucleotide present in thecomposition that is needed to provide the desired level of BDNFexpression in the subject to be treated to give the anticipatedphysiological response. The term “physiologically effective amount” isthat amount delivered to a subject to give the desired palliative orcurative effect. The term “pharmaceutically acceptable carrier” meansthat the carrier can be administered to a subject with no significantadverse toxicological effects to the subject.

The single stranded oligonucleotide molecules of the invention can beincorporated into pharmaceutical compositions suitable foradministration. Such compositions typically include one or more speciesof single stranded oligonucleotide and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active compound,use thereof in the compositions is contemplated. Supplementary activecompounds can also be incorporated into the compositions.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, or intrathecal or intraventricularadministration.

The route and site of administration may be chosen to enhance targeting.For example, to target muscle cells, intramuscular injection into themuscles of interest would be a logical choice. Lung cells might betargeted by administering the single stranded oligonucleotide in aerosolform. The vascular endothelial cells could be targeted by coating aballoon catheter with the single stranded oligonucleotide andmechanically introducing the oligonucleotide.

Topical administration refers to the delivery to a subject by contactingthe formulation directly to a surface of the subject. The most commonform of topical delivery is to the skin, but a composition disclosedherein can also be directly applied to other surfaces of the body, e.g.,to the eye, a mucous membrane, to surfaces of a body cavity or to aninternal surface. As mentioned above, the most common topical deliveryis to the skin. The term encompasses several routes of administrationincluding, but not limited to, topical and transdermal. These modes ofadministration typically include penetration of the skin's permeabilitybarrier and efficient delivery to the target tissue or stratum. Topicaladministration can be used as a means to penetrate the epidermis anddermis and ultimately achieve systemic delivery of the composition.Topical administration can also be used as a means to selectivelydeliver oligonucleotides to the epidermis or dermis of a subject, or tospecific strata thereof, or to an underlying tissue.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Transdermal delivery is a valuable route for the administration of lipidsoluble therapeutics. The dermis is more permeable than the epidermisand therefore absorption is much more rapid through abraded, burned ordenuded skin. Inflammation and other physiologic conditions thatincrease blood flow to the skin also enhance transdermal adsorption.Absorption via this route may be enhanced by the use of an oily vehicle(inunction) or through the use of one or more penetration enhancers.Other effective ways to deliver a composition disclosed herein via thetransdermal route include hydration of the skin and the use ofcontrolled release topical patches. The transdermal route provides apotentially effective means to deliver a composition disclosed hereinfor systemic and/or local therapy. In addition, iontophoresis (transferof ionic solutes through biological membranes under the influence of anelectric field), phonophoresis or sonophoresis (use of ultrasound toenhance the absorption of various therapeutic agents across biologicalmembranes, notably the skin and the cornea), and optimization of vehiclecharacteristics relative to dose position and retention at the site ofadministration may be useful methods for enhancing the transport oftopically applied compositions across skin and mucosal sites.

Both the oral and nasal membranes offer advantages over other routes ofadministration. For example, oligonucleotides administered through thesemembranes may have a rapid onset of action, provide therapeutic plasmalevels, avoid first pass effect of hepatic metabolism, and avoidexposure of the oligonucleotides to the hostile gastrointestinal (GI)environment. Additional advantages include easy access to the membranesites so that the oligonucleotide can be applied, localized and removedeasily.

In oral delivery, compositions can be targeted to a surface of the oralcavity, e.g., to sublingual mucosa which includes the membrane ofventral surface of the tongue and the floor of the mouth or the buccalmucosa which constitutes the lining of the cheek. The sublingual mucosais relatively permeable thus giving rapid absorption and acceptablebioavailability of many agents. Further, the sublingual mucosa isconvenient, acceptable and easily accessible.

A pharmaceutical composition of single stranded oligonucleotide may alsobe administered to the buccal cavity of a human being by spraying intothe cavity, without inhalation, from a metered dose spray dispenser, amixed micellar pharmaceutical formulation as described above and apropellant. In one embodiment, the dispenser is first shaken prior tospraying the pharmaceutical formulation and propellant into the buccalcavity.

Compositions for oral administration include powders or granules,suspensions or solutions in water, syrups, slurries, emulsions, elixirsor non-aqueous media, tablets, capsules, lozenges, or troches. In thecase of tablets, carriers that can be used include lactose, sodiumcitrate and salts of phosphoric acid. Various disintegrants such asstarch, and lubricating agents such as magnesium stearate, sodium laurylsulfate and talc, are commonly used in tablets. For oral administrationin capsule form, useful diluents are lactose and high molecular weightpolyethylene glycols. When aqueous suspensions are required for oraluse, the nucleic acid compositions can be combined with emulsifying andsuspending agents. If desired, certain sweetening and/or flavoringagents can be added.

Parenteral administration includes intravenous drip, subcutaneous,intraperitoneal or intramuscular injection, intrathecal orintraventricular administration. In some embodiments, parentaladministration involves administration directly to the site of disease(e.g. injection into a tumor).

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

Any of the single stranded oligonucleotides described herein can beadministered to ocular tissue. For example, the compositions can beapplied to the surface of the eye or nearby tissue, e.g., the inside ofthe eyelid. For ocular administration, ointments or droppable liquidsmay be delivered by ocular delivery systems known to the art such asapplicators or eye droppers. Such compositions can include mucomimeticssuch as hyaluronic acid, chondroitin sulfate, hydroxypropylmethylcellulose or poly(vinyl alcohol), preservatives such as sorbicacid, EDTA or benzylchronium chloride, and the usual quantities ofdiluents and/or carriers. The single stranded oligonucleotide can alsobe administered to the interior of the eye, and can be introduced by aneedle or other delivery device which can introduce it to a selectedarea or structure.

Pulmonary delivery compositions can be delivered by inhalation by thepatient of a dispersion so that the composition, preferably singlestranded oligonucleotides, within the dispersion can reach the lungwhere it can be readily absorbed through the alveolar region directlyinto blood circulation. Pulmonary delivery can be effective both forsystemic delivery and for localized delivery to treat diseases of thelungs.

Pulmonary delivery can be achieved by different approaches, includingthe use of nebulized, aerosolized, micellular and dry powder-basedformulations. Delivery can be achieved with liquid nebulizers,aerosol-based inhalers, and dry powder dispersion devices. Metered-dosedevices are preferred. One of the benefits of using an atomizer orinhaler is that the potential for contamination is minimized because thedevices are self-contained. Dry powder dispersion devices, for example,deliver agents that may be readily formulated as dry powders. A singlestranded oligonucleotide composition may be stably stored as lyophilizedor spray-dried powders by itself or in combination with suitable powdercarriers. The delivery of a composition for inhalation can be mediatedby a dosing timing element which can include a timer, a dose counter,time measuring device, or a time indicator which when incorporated intothe device enables dose tracking, compliance monitoring, and/or dosetriggering to a patient during administration of the aerosol medicament.

The term “powder” means a composition that consists of finely dispersedsolid particles that are free flowing and capable of being readilydispersed in an inhalation device and subsequently inhaled by a subjectso that the particles reach the lungs to permit penetration into thealveoli. Thus, the powder is said to be “respirable.” Preferably theaverage particle size is less than about 10 μm in diameter preferablywith a relatively uniform spheroidal shape distribution. More preferablythe diameter is less than about 7.5 μm and most preferably less thanabout 5.0 μm. Usually the particle size distribution is between about0.1 μm and about 5 μm in diameter, particularly about 0.3 μm to about 5μm.

The term “dry” means that the composition has a moisture content belowabout 10% by weight (% w) water, usually below about 5% w and preferablyless it than about 3% w. A dry composition can be such that theparticles are readily dispersible in an inhalation device to form anaerosol.

The types of pharmaceutical excipients that are useful as carrierinclude stabilizers such as human serum albumin (HSA), bulking agentssuch as carbohydrates, amino acids and polypeptides; pH adjusters orbuffers; salts such as sodium chloride; and the like. These carriers maybe in a crystalline or amorphous form or may be a mixture of the two.

Suitable pH adjusters or buffers include organic salts prepared fromorganic acids and bases, such as sodium citrate, sodium ascorbate, andthe like; sodium citrate is preferred. Pulmonary administration of amicellar single stranded oligonucleotide formulation may be achievedthrough metered dose spray devices with propellants such astetrafluoroethane, heptafluoroethane, dimethylfluoropropane,tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFCand CFC propellants.

Exemplary devices include devices which are introduced into thevasculature, e.g., devices inserted into the lumen of a vascular tissue,or which devices themselves form a part of the vasculature, includingstents, catheters, heart valves, and other vascular devices. Thesedevices, e.g., catheters or stents, can be placed in the vasculature ofthe lung, heart, or leg.

Other devices include non-vascular devices, e.g., devices implanted inthe peritoneum, or in organ or glandular tissue, e.g., artificialorgans. The device can release a therapeutic substance in addition to asingle stranded oligonucleotide, e.g., a device can release insulin.

In one embodiment, unit doses or measured doses of a composition thatincludes single stranded oligonucleotide are dispensed by an implanteddevice. The device can include a sensor that monitors a parameter withina subject. For example, the device can include pump, e.g., and,optionally, associated electronics.

Tissue, e.g., cells or organs can be treated with a single strandedoligonucleotide, ex vivo and then administered or implanted in asubject. The tissue can be autologous, allogeneic, or xenogeneic tissue.E.g., tissue can be treated to reduce graft v. host disease. In otherembodiments, the tissue is allogeneic and the tissue is treated to treata disorder characterized by unwanted gene expression in that tissue.E.g., tissue, e.g., hematopoietic cells, e.g., bone marrow hematopoieticcells, can be treated to inhibit unwanted cell proliferation.Introduction of treated tissue, whether autologous or transplant, can becombined with other therapies. In some implementations, the singlestranded oligonucleotide treated cells are insulated from other cells,e.g., by a semi-permeable porous barrier that prevents the cells fromleaving the implant, but enables molecules from the body to reach thecells and molecules produced by the cells to enter the body. In oneembodiment, the porous barrier is formed from alginate.

In one embodiment, a contraceptive device is coated with or contains asingle stranded oligonucleotide. Exemplary devices include condoms,diaphragms, IUD (implantable uterine devices, sponges, vaginal sheaths,and birth control devices.

Dosage

In one aspect, the invention features a method of administering a singlestranded oligonucleotide (e.g., as a compound or as a component of acomposition) to a subject (e.g., a human subject). In one embodiment,the unit dose is between about 10 mg and 25 mg per kg of bodyweight. Inone embodiment, the unit dose is between about 1 mg and 100 mg per kg ofbodyweight. In one embodiment, the unit dose is between about 0.1 mg and500 mg per kg of bodyweight. In some embodiments, the unit dose is morethan 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 25, 50 or 100 mgper kg of bodyweight.

The defined amount can be an amount effective to treat or prevent adisease or disorder, e.g., a disease or disorder associated with theBDNF. The unit dose, for example, can be administered by injection(e.g., intravenous or intramuscular), an inhaled dose, or a topicalapplication.

In some embodiments, the unit dose is administered daily. In someembodiments, less frequently than once a day, e.g., less than every 2,4, 8 or 30 days. In another embodiment, the unit dose is notadministered with a frequency (e.g., not a regular frequency). Forexample, the unit dose may be administered a single time. In someembodiments, the unit dose is administered more than once a day, e.g.,once an hour, two hours, four hours, eight hours, twelve hours, etc.

In one embodiment, a subject is administered an initial dose and one ormore maintenance doses of a single stranded oligonucleotide. Themaintenance dose or doses are generally lower than the initial dose,e.g., one-half less of the initial dose. A maintenance regimen caninclude treating the subject with a dose or doses ranging from 0.0001 to100 mg/kg of body weight per day, e.g., 100, 10, 1, 0.1, 0.01, 0.001, or0.0001 mg per kg of bodyweight per day. The maintenance doses may beadministered no more than once every 1, 5, 10, or 30 days. Further, thetreatment regimen may last for a period of time which will varydepending upon the nature of the particular disease, its severity andthe overall condition of the patient. In some embodiments the dosage maybe delivered no more than once per day, e.g., no more than once per 24,36, 48, or more hours, e.g., no more than once for every 5 or 8 days.Following treatment, the patient can be monitored for changes in hiscondition and for alleviation of the symptoms of the disease state. Thedosage of the oligonucleotide may either be increased in the event thepatient does not respond significantly to current dosage levels, or thedose may be decreased if an alleviation of the symptoms of the diseasestate is observed, if the disease state has been ablated, or ifundesired side-effects are observed.

The effective dose can be administered in a single dose or in two ormore doses, as desired or considered appropriate under the specificcircumstances. If desired to facilitate repeated or frequent infusions,implantation of a delivery device, e.g., a pump, semi-permanent stent(e.g., intravenous, intraperitoneal, intracisternal or intracapsular),or reservoir may be advisable.

In some embodiments, the oligonucleotide pharmaceutical compositionincludes a plurality of single stranded oligonucleotide species. Inanother embodiment, the single stranded oligonucleotide species hassequences that are non-overlapping and non-adjacent to another specieswith respect to a naturally occurring target sequence (e.g., aPRC2-associated region). In another embodiment, the plurality of singlestranded oligonucleotide species is specific for differentPRC2-associated regions. In another embodiment, the single strandedoligonucleotide is allele specific. In some cases, a patient is treatedwith a single stranded oligonucleotide in conjunction with othertherapeutic modalities.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound of the invention is administered inmaintenance doses, ranging from 0.0001 mg to 100 mg per kg of bodyweight.

The concentration of the single stranded oligonucleotide composition isan amount sufficient to be effective in treating or preventing adisorder or to regulate a physiological condition in humans. Theconcentration or amount of single stranded oligonucleotide administeredwill depend on the parameters determined for the agent and the method ofadministration, e.g. nasal, buccal, pulmonary. For example, nasalformulations may tend to require much lower concentrations of someingredients in order to avoid irritation or burning of the nasalpassages. It is sometimes desirable to dilute an oral formulation up to10-100 times in order to provide a suitable nasal formulation.

Certain factors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a single strandedoligonucleotide can include a single treatment or, preferably, caninclude a series of treatments. It will also be appreciated that theeffective dosage of a single stranded oligonucleotide used for treatmentmay increase or decrease over the course of a particular treatment. Forexample, the subject can be monitored after administering a singlestranded oligonucleotide composition. Based on information from themonitoring, an additional amount of the single stranded oligonucleotidecomposition can be administered.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of BDNF expression levels in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual compounds, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models. In some embodiments, the animal modelsinclude transgenic animals that express a human BDNF. In anotherembodiment, the composition for testing includes a single strandedoligonucleotide that is complementary, at least in an internal region,to a sequence that is conserved between BDNF in the animal model and theBDNF in a human.

In one embodiment, the administration of the single strandedoligonucleotide composition is parenteral, e.g. intravenous (e.g., as abolus or as a diffusible infusion), intradermal, intraperitoneal,intramuscular, intrathecal, intraventricular, intracranial,subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal,oral, vaginal, topical, pulmonary, intranasal, urethral or ocular.Administration can be provided by the subject or by another person,e.g., a health care provider. The composition can be provided inmeasured doses or in a dispenser which delivers a metered dose. Selectedmodes of delivery are discussed in more detail below.

Kits

In certain aspects of the invention, kits are provided, comprising acontainer housing a composition comprising a single strandedoligonucleotide. In some embodiments, the composition is apharmaceutical composition comprising a single stranded oligonucleotideand a pharmaceutically acceptable carrier. In some embodiments, theindividual components of the pharmaceutical composition may be providedin one container. Alternatively, it may be desirable to provide thecomponents of the pharmaceutical composition separately in two or morecontainers, e.g., one container for single stranded oligonucleotides,and at least another for a carrier compound. The kit may be packaged ina number of different configurations such as one or more containers in asingle box. The different components can be combined, e.g., according toinstructions provided with the kit. The components can be combinedaccording to a method described herein, e.g., to prepare and administera pharmaceutical composition. The kit can also include a deliverydevice.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Materials and Methods: Real Time PCR

RNA was harvested from the cells using Promega SV 96 Total RNA Isolationsystem or Trizol omitting the DNAse step. In separate pilot experiments,50 ng of RNA was determined to be sufficient template for the reversetranscriptase reaction. RNA harvested from cells was normalized so that50 ng of RNA was input to each reverse transcription reaction. For thefew samples that were too dilute to reach this limit, the maximum inputvolume was added. Reverse transcriptase reaction was performed using theSuperscript II kit and real time PCR performed on cDNA samples usingicycler SYBR green chemistry (Biorad). A baseline level of mRNAexpression for each target gene was determined through quantitative PCRas outlined above. Baseline levels were also determined for mRNA ofvarious housekeeping genes which are constitutively expressed. A“control” housekeeping gene with approximately the same level ofbaseline expression as the target gene was chosen for comparisonpurposes.

Cell Culture

Human hepatocyte Hep3B, human hepatocyte HepG2 cells, mouse hepatomaHepa1-6 cells, and human renal proximal tubule epithelial cells (RPTEC)were cultured using conditions known in the art (see, e.g. CurrentProtocols in Cell Biology). Details of the cell lines used in theexperiments described herein are provided in Table 5.

TABLE 5 Cell lines Culture Cell line Source Species Gender Cell TypeTissue Status Conditions Hep3B ATCC human M hepatocytes liverimmortalized Eagle's MEM + 10% FBS liv mus from C57BI/6 mouse N/Amultiple cell liver primary C57BI mice (mice types obtained from JacksonLabs) kid mus from C57BI/6 mouse N/A multiple cell kidney primary C57BImice (mice types obtained from Jackson Labs)

Oligonucleotide Design

Oligonucleotides were designed within PRC2-interacting regions in orderto upregulate BDNF. The sequence and structure of each oligonucleotideis shown in Table 4. The following table provides a description of thenucleotide analogs, modifications and intranucleotide linkages used forcertain oligonucleotides tested and described in Table 3.

TABLE 3 Oligonucleotide Modifications Symbol Feature Description bio 5′biotin dAs DNA w/3′ thiophosphate dCs DNA w/3′ thiophosphate dGs DNAw/3′ thiophosphate dTs DNA w/3′ thiophosphate dG DNA enaAs ENA w/3′thiophosphate enaCs ENA w/3′ thiophosphate enaGs ENA w/3′ thiophosphateenaTs ENA w/3′ thiophosphate fluAs 2′-fluoro w/3′ thiophosphate fluCs2′-fluoro w/3′ thiophosphate fluGs 2′-fluoro w/3′ thiophosphate fluUs2′-fluoro w/3′ thiophosphate lnaAs LNA w/3′ thiophosphate lnaCs LNA w/3′thiophosphate lnaGs LNA w/3′ thiophosphate lnaTs LNA w/3′ thiophosphateomeAs 2′-OMe w/3′ thiophosphate omeCs 2′-OMe w/3′ thiophosphate omeGs2′-OMe w/3′ thiophosphate omeTs 2′-OMe w/3′ thiophosphate lnaAs-Sup LNAw/3′ thiophosphate at 3′ terminus lnaCs-Sup LNA w/3′ thiophosphate at 3′terminus lnaGs-Sup LNA w/3′ thiophosphate at 3′ terminus lnaTs-Sup LNAw/3′ thiophosphate at 3′ terminus lnaA-Sup LNA w/3′ OH at 3′ terminuslnaC-Sup LNA w/3′ OH at 3′ terminus lnaG-Sup LNA w/3′ OH at 3′ terminuslnaT-Sup LNA w/3′ OH at 3′ terminus omeA-Sup 2′-OMe w/3′ OH at 3′terminus omeC-Sup 2′-OMe w/3′ OH at 3′ terminus omeG-Sup 2′-OMe w/3′ OHat 3′ terminus omeU-Sup 2′-OMe w/3′ OH at 3′ terminus dAs-Sup DNA w/3′thiophosphate at 3′ terminus dCs-Sup DNA w/3′ thiophosphate at 3′terminus dGs-Sup DNA w/3′ thiophosphate at 3′ terminus dTs-Sup DNA w/3′thiophosphate at 3′ terminus dA-Sup DNA w/3′ OH at 3′ terminus dC-SupDNA w/3′ OH at 3′ terminus dG-Sup DNA w/3′ OH at 3′ terminus dT-Sup DNAw/3′ OH at 3′ terminusIn Vitro Transfection of Cells with Oligonucleotides

Cells were seeded into each well of 24-well plates at a density of25,000 cells per 500 uL and transfections were performed withLipofectamine and the single stranded oligonucleotides. Control wellscontained Lipofectamine alone. At 48 hours post-transfection,approximately 200 uL of cell culture supernatants were stored at −80 C.for ELISA. At 48 hours post-transfection, RNA was harvested from thecells and quantitative PCR was carried out as outlined above. Thepercent induction of target mRNA expression by each oligonucleotide wasdetermined by normalizing mRNA levels in the presence of theoligonucleotide to the mRNA levels in the presence of control(Lipofectamine alone). This was compared side-by-side with the increasein mRNA expression of the “control” housekeeping gene.

Results: In Vitro Delivery of Single Stranded OligonucleotidesUpregulated BDNF Expression

Oligonucleotides were designed as candidates for upregulating BDNFexpression. A total of 40 single stranded oligonucleotides were designedto be complementary to a PRC2-interacting region within a sequence asset forth in SEQ ID NO: 1 or 2. 30 of the oligonucleotides were testedin at least duplicate. The sequence and structural features of theoligonucleotides are set forth in Table 2. Briefly, cells weretransfected in vitro with each of the 30 oligonucleotides as describedabove. BDNF expression in cells following treatment was evaluated byqRT-PCR. Oligonucleotides that upregulated BDNF expression wereidentified. Further details are outlined in Table 2.

Tables

TABLE 1 Hexamers that are not seed sequences of human miRNAsAAAAAA, AAAAAG, AAAACA, AAAAGA, AAAAGC, AAAAGG, AAAAUA, AAACAA, AAACAC, AAACAG, AAACAU, AAACCC, AAACCU, AAACGA, AAACGC, AAACGU, AAACUA, AAACUC, AAACUU, AAAGAU, AAAGCC, AAAGGA, AAAGGG, AAAGUC, AAAUAC, AAAUAU, AAAUCG, AAAUCU, AAAUGC, AAAUGU, AAAUUA, AAAUUG, AACAAC, AACAAG, AACAAU, AACACA, AACACG, AACAGA, AACAGC, AACAGG, AACAUC, AACAUG, AACCAA, AACCAC, AACCAG, AACCAU, AACCCC, AACCCG, AACCGA, AACCGC, AACCGG, AACCUA, AACCUU, AACGAA, AACGAC, AACGAG, AACGAU, AACGCU, AACGGG, AACGGU, AACGUA, AACGUC, AACGUG, AACGUU, AACUAU, AACUCA, AACUCC, AACUCG, AACUGA, AACUGC, AACUGU, AACUUA, AACUUC, AACUUG, AACUUU, AAGAAA, AAGAAG, AAGAAU, AAGACG, AAGAGA, AAGAGC, AAGAGG, AAGAGU, AAGAUU, AAGCAA, AAGCAC, AAGCAG, AAGCAU, AAGCCA, AAGCCC, AAGCCG, AAGCCU, AAGCGA, AAGCGG, AAGCGU, AAGCUA, AAGGAA, AAGGAC, AAGGCU, AAGGGC, AAGGGU, AAGGUU, AAGUAA, AAGUAC, AAGUAU, AAGUCC, AAGUCG, AAGUGA, AAGUGG, AAGUUA, AAGUUU, AAUAAA, AAUAAC, AAUAAG, AAUAAU, AAUACA, AAUACC, AAUACG, AAUAGA, AAUAGC, AAUAGG, AAUAGU, AAUAUC, AAUAUU, AAUCAA, AAUCAU, AAUCCA, AAUCCC, AAUCCG, AAUCGA, AAUCGC, AAUCGU, AAUCUA, AAUCUG, AAUCUU, AAUGAA, AAUGAC, AAUGAG, AAUGAU, AAUGCG, AAUGCU, AAUGGA, AAUGGU, AAUGUA, AAUGUC, AAUGUG, AAUUAA, AAUUAC, AAUUAG, AAUUCC, AAUUCG, AAUUGA, AAUUGG, AAUUGU, AAUUUC, AAUUUG, ACAAAA, ACAAAC, ACAAAG, ACAAAU, ACAACC, ACAACG, ACAACU, ACAAGA, ACAAGC, ACAAGU, ACAAUC, ACAAUG, ACAAUU, ACACAG, ACACCA, ACACCC, ACACCG, ACACCU, ACACGA, ACACGC, ACACGU, ACACUC, ACACUG, ACACUU, ACAGAA, ACAGAC, ACAGCC, ACAGCG, ACAGCU, ACAGGG, ACAGUC, ACAGUG, ACAGUU, ACAUAA, ACAUAC, ACAUCC, ACAUCG, ACAUCU, ACAUGA, ACAUGC, ACAUGU, ACAUUG, ACAUUU, ACCAAA, ACCAAC, ACCAAG, ACCAAU, ACCACC, ACCACG, ACCAGA, ACCAGU, ACCAUA, ACCAUG, ACCAUU, ACCCAA, ACCCAC, ACCCCA, ACCCCG, ACCCGA, ACCCGC, ACCCUA, ACCCUC, ACCCUU, ACCGAA, ACCGAC, ACCGAU, ACCGCA, ACCGCC, ACCGCG, ACCGCU, ACCGGA, ACCGGC, ACCGGU, ACCGUA, ACCGUC, ACCGUG, ACCGUU, ACCUAA, ACCUAC, ACCUAG, ACCUAU, ACCUCA, ACCUCC, ACCUCG, ACCUCU, ACCUGA, ACCUGC, ACCUGU, ACCUUA, ACCUUC, ACCUUU, ACGAAA, ACGAAC, ACGAAG, ACGAAU, ACGACA, ACGACC, ACGACG, ACGACU, ACGAGA, ACGAGC, ACGAGG, ACGAGU, ACGAUA, ACGAUC, ACGAUG, ACGAUU, ACGCAA, ACGCAG, ACGCAU, ACGCCC, ACGCCG, ACGCCU, ACGCGA, ACGCGG, ACGCGU, ACGCUA, ACGCUG, ACGCUU, ACGGAA, ACGGAC, ACGGAG, ACGGAU, ACGGCC, ACGGCG, ACGGCU, ACGGGC, ACGGGG, ACGGGU, ACGGUA, ACGGUC, ACGGUG, ACGGUU, ACGUAA, ACGUAC, ACGUAU, ACGUCC, ACGUCG, ACGUCU, ACGUGA, ACGUGC, ACGUGG, ACGUGU, ACGUUA, ACGUUC, ACGUUG, ACGUUU, ACUAAA, ACUAAG, ACUAAU, ACUACA, ACUACC, ACUACG, ACUACU, ACUAGG, ACUAUC, ACUAUG, ACUAUU, ACUCAU, ACUCCC, ACUCCG, ACUCCU, ACUCGA, ACUCGC, ACUCGG, ACUCUC, ACUCUU, ACUGAG, ACUGAU, ACUGCC, ACUGCG, ACUGCU, ACUGGG, ACUGGU, ACUGUC, ACUUAA, ACUUAC, ACUUAU, ACUUCA, ACUUCC, ACUUCG, ACUUCU, ACUUGA, ACUUGC, ACUUGU, ACUUUA, ACUUUC, ACUUUG, AGAAAA, AGAAAC, AGAAAG, AGAACC, AGAACG, AGAACU, AGAAGC, AGAAGU, AGAAUA, AGAAUC, AGAAUG, AGAAUU, AGACAA, AGACAC, AGACAU, AGACCA, AGACCC, AGACCG, AGACCU, AGACGA, AGACGC, AGACGU, AGACUA, AGACUC, AGACUU, AGAGAC, AGAGAG, AGAGAU, AGAGCC, AGAGCG, AGAGCU, AGAGGC, AGAGGG, AGAGGU, AGAGUA, AGAGUU, AGAUAC, AGAUAG, AGAUAU, AGAUCC, AGAUCG, AGAUCU, AGAUGA, AGAUGC, AGAUGG, AGAUUA, AGAUUC, AGAUUG, AGAUUU, AGCAAC, AGCACA, AGCACG, AGCACU, AGCAGA, AGCAUA, AGCAUC, AGCAUG, AGCCAA, AGCCAU, AGCCCA, AGCCGA, AGCCGC, AGCCGG, AGCCGU, AGCCUA, AGCCUC, AGCGAA, AGCGAG, AGCGAU, AGCGCA, AGCGCC, AGCGCG, AGCGCU, AGCGGA, AGCGGC, AGCGGU, AGCGUA, AGCGUC, AGCGUG, AGCGUU, AGCUAA, AGCUAC, AGCUAG, AGCUAU, AGCUCA, AGCUCC, AGCUCG, AGCUCU, AGCUGA, AGCUGG, AGCUGU, AGCUUC, AGCUUU, AGGAAU, AGGACC, AGGACG, AGGAGA, AGGAGU, AGGAUA, AGGCAA, AGGCAU, AGGCCG, AGGCGA, AGGCGC, AGGCGG, AGGCUA, AGGCUC, AGGCUU, AGGGAC, AGGGAU, AGGGGA, AGGGGU, AGGGUA, AGGGUG, AGGUAA, AGGUAC, AGGUCA, AGGUCC, AGGUCU, AGGUGA, AGGUGC, AGGUGG, AGGUGU, AGGUUC, AGGUUG, AGUAAA, AGUAAG, AGUAAU, AGUACA, AGUACG, AGUAGC, AGUAGG, AGUAUA, AGUAUC, AGUAUG, AGUAUU, AGUCAA, AGUCAC, AGUCAG, AGUCAU, AGUCCA, AGUCCG, AGUCCU, AGUCGA, AGUCGC, AGUCGG, AGUCGU, AGUCUA, AGUCUC, AGUCUG, AGUCUU, AGUGAA, AGUGAC, AGUGCG, AGUGGG, AGUGUC, AGUUAA, AGUUAC, AGUUAG, AGUUCC, AGUUCG, AGUUGA, AGUUGC, AGUUGU, AGUUUA, AGUUUC, AGUUUG, AGUUUU, AUAAAC, AUAAAU, AUAACA, AUAACC, AUAACG, AUAACU, AUAAGA, AUAAGC, AUAAGG, AUAAGU, AUAAUC, AUAAUG, AUAAUU, AUACAC, AUACAG, AUACAU, AUACCA, AUACCC, AUACCG, AUACGA, AUACGC, AUACGG, AUACGU, AUACUA, AUACUC, AUACUG, AUACUU, AUAGAA, AUAGAC, AUAGAU, AUAGCA, AUAGCG, AUAGCU, AUAGGA, AUAGGU, AUAGUA, AUAGUC, AUAGUG, AUAGUU, AUAUAC, AUAUAG, AUAUCC, AUAUCG, AUAUCU, AUAUGA, AUAUGC, AUAUGG, AUAUGU, AUAUUC, AUAUUG, AUAUUU, AUCAAA, AUCAAC, AUCAAG, AUCAAU, AUCACA, AUCACC, AUCACG, AUCAGC, AUCAGG, AUCCAA, AUCCAU, AUCCCC, AUCCCG, AUCCGA, AUCCGC, AUCCGG, AUCCUA, AUCCUC, AUCCUG, AUCGAA, AUCGAC, AUCGAG, AUCGAU, AUCGCA, AUCGCC, AUCGCG, AUCGCU, AUCGGC, AUCGGG, AUCGGU, AUCGUC, AUCGUG, AUCGUU, AUCUAA, AUCUAC, AUCUAG, AUCUAU, AUCUCC, AUCUCG, AUCUGU, AUCUUG, AUCUUU, AUGAAA, AUGAAC, AUGAAG, AUGAAU, AUGACC, AUGACU, AUGAGG, AUGAGU, AUGAUA, AUGAUC, AUGAUU, AUGCAA, AUGCAG, AUGCCA, AUGCCC, AUGCCG, AUGCGA, AUGCGG, AUGCGU, AUGCUC, AUGCUU, AUGGAC, AUGGCC, AUGGGA, AUGGGC, AUGGGU, AUGGUC, AUGGUG, AUGUAC, AUGUAU, AUGUCA, AUGUCC, AUGUCG, AUGUGU, AUGUUA, AUGUUC, AUUAAA, AUUAAC, AUUAAG, AUUAAU, AUUACA, AUUACC, AUUACG, AUUACU, AUUAGA, AUUAGC, AUUAGG, AUUAGU, AUUAUA, AUUAUC, AUUAUG, AUUCAC, AUUCCA, AUUCCG, AUUCCU, AUUCGA, AUUCGC, AUUCGG, AUUCGU, AUUCUA, AUUCUC, AUUCUU, AUUGAA, AUUGAC, AUUGAU, AUUGCC, AUUGCG, AUUGCU, AUUGGA, AUUGGC, AUUGGG, AUUGGU, AUUGUA, AUUGUC, AUUGUG, AUUGUU, AUUUAA, AUUUAG, AUUUAU, AUUUCC, AUUUCG, AUUUCU, AUUUGA, AUUUGC, AUUUGU, AUUUUA, AUUUUC, AUUUUG, AUUUUU, CAAAAG, CAAACA, CAAACC, CAAACG, CAAACU, CAAAGA, CAAAGG, CAAAUA, CAAAUU, CAACAC, CAACAU, CAACCA, CAACCC, CAACCG, CAACGA, CAACGC, CAACGG, CAACGU, CAACUA, CAACUC, CAACUG, CAACUU, CAAGAA, CAAGAC, CAAGAU, CAAGCA, CAAGCC, CAAGCG, CAAGCU, CAAGGA, CAAGGG, CAAGUC, CAAGUG, CAAGUU, CAAUAA, CAAUAC, CAAUAG, CAAUCC, CAAUCG, CAAUCU, CAAUGA, CAAUGC, CAAUGG, CAAUGU, CAAUUC, CAAUUG, CAAUUU, CACAAU, CACACA, CACACG, CACACU, CACAGA, CACAGC, CACAGG, CACAUA, CACAUC, CACAUU, CACCAA, CACCAC, CACCAU, CACCCA, CACCCC, CACCCG, CACCGA, CACCGC, CACCGG, CACCGU, CACCUA, CACCUU, CACGAA, CACGAC, CACGAG, CACGAU, CACGCA, CACGCC, CACGCU, CACGGA, CACGGC, CACGGG, CACGGU, CACGUA, CACGUC, CACGUG, CACGUU, CACUAA, CACUAG, CACUAU, CACUCA, CACUCG, CACUGA, CACUGC, CACUGG, CACUUA, CACUUC, CACUUU, CAGAAA, CAGAAG, CAGAAU, CAGACC, CAGACG, CAGAGC, CAGAUA, CAGAUC, CAGCCG, CAGCCU, CAGCGA, CAGCGC, CAGCGG, CAGCGU, CAGCUC, CAGCUU, CAGGAU, CAGGGG, CAGGGU, CAGGUA, CAGGUC, CAGGUU, CAGUAC, CAGUCG, CAGUUG, CAUAAA, CAUAAC, CAUAAG, CAUAAU, CAUACA, CAUACC, CAUACG, CAUACU, CAUAGA, CAUAGG, CAUAGU, CAUAUA, CAUAUC, CAUAUG, CAUCAA, CAUCAC, CAUCAG, CAUCAU, CAUCCA, CAUCCC, CAUCCG, CAUCGA, CAUCGC, CAUCGG, CAUCGU, CAUCUA, CAUCUC, CAUCUG, CAUCUU, CAUGAA, CAUGAC, CAUGAG, CAUGAU, CAUGCA, CAUGCC, CAUGCG, CAUGCU, CAUGGC, CAUGGG, CAUGGU, CAUGUA, CAUGUC, CAUGUU, CAUUAA, CAUUAC, CAUUAG, CAUUCA, CAUUCC, CAUUCG, CAUUCU, CAUUGA, CAUUGG, CAUUUC, CAUUUG, CAUUUU, CCAAAA, CCAAAC, CCAAAG, CCAAAU, CCAACA, CCAACC, CCAACG, CCAACU, CCAAGA, CCAAGC, CCAAGG, CCAAUC, CCAAUG, CCAAUU, CCACAA, CCACAC, CCACAG, CCACAU, CCACCA, CCACCC, CCACCG, CCACCU, CCACGA, CCACGC, CCACGG, CCACGU, CCACUA, CCACUC, CCACUU, CCAGAA, CCAGAC, CCAGAG, CCAGCC, CCAGGU, CCAGUC, CCAGUU, CCAUAA, CCAUAC, CCAUAG, CCAUAU, CCAUCA, CCAUCC, CCAUCU, CCAUGA, CCAUGC, CCAUGG, CCAUUC, CCAUUG, CCAUUU, CCCAAC, CCCAAG, CCCAAU, CCCACA, CCCAGA, CCCAGC, CCCAGU, CCCAUA, CCCAUC, CCCAUG, CCCAUU, CCCCAA, CCCCAG, CCCCAU, CCCCCC, CCCCCG, CCCCCU, CCCCGA, CCCCGC, CCCCGU, CCCCUA, CCCCUC, CCCGAA, CCCGAC, CCCGAU, CCCGCA, CCCGCU, CCCGGA, CCCGGC, CCCGUA, CCCGUG, CCCGUU, CCCUAA, CCCUAG, CCCUCA, CCCUCU, CCCUGC, CCCUUA, CCCUUC, CCCUUU, CCGAAA, CCGAAC, CCGAAU, CCGACA, CCGACC, CCGACG, CCGACU, CCGAGA, CCGAGG, CCGAGU, CCGAUA, CCGAUC, CCGAUG, CCGAUU, CCGCAA, CCGCAC, CCGCAG, CCGCAU, CCGCCA, CCGCCC, CCGCCG, CCGCCU, CCGCGA, CCGCGC, CCGCGG, CCGCGU, CCGCUA, CCGCUC, CCGCUG, CCGCUU, CCGGAA, CCGGAU, CCGGCA, CCGGCC, CCGGCG, CCGGCU, CCGGGA, CCGGGC, CCGGGG, CCGGGU, CCGGUA, CCGGUC, CCGGUG, CCGUAA, CCGUAG, CCGUAU, CCGUCA, CCGUCC, CCGUCG, CCGUGA, CCGUGU, CCGUUA, CCGUUC, CCGUUG, CCGUUU, CCUAAC, CCUAAG, CCUAAU, CCUACA, CCUACC, CCUACG, CCUACU, CCUAGA, CCUAGC, CCUAGG, CCUAGU, CCUAUA, CCUAUC, CCUAUG, CCUAUU, CCUCAA, CCUCAC, CCUCAG, CCUCAU, CCUCCA, CCUCCC, CCUCCG, CCUCGA, CCUCGC, CCUCGG, CCUCGU, CCUCUA, CCUCUG, CCUGAC, CCUGAU, CCUGCA, CCUGGG, CCUGGU, CCUGUU, CCUUAA, CCUUAC, CCUUAG, CCUUAU, CCUUCG, CCUUGA, CCUUGU, CCUUUA, CCUUUC, CCUUUU, CGAAAA, CGAAAC, CGAAAG, CGAAAU, CGAACA, CGAACC, CGAACG, CGAACU, CGAAGA, CGAAGC, CGAAGG, CGAAGU, CGAAUA, CGAAUC, CGAAUG, CGAAUU, CGACAA, CGACAC, CGACAU, CGACCA, CGACCU, CGACGA, CGACGC, CGACGG, CGACGU, CGACUA, CGACUG, CGACUU, CGAGAA, CGAGAC, CGAGAG, CGAGAU, CGAGCA, CGAGCC, CGAGCG, CGAGCU, CGAGGC, CGAGGG, CGAGGU, CGAGUA, CGAGUC, CGAGUG, CGAGUU, CGAUAA, CGAUAC, CGAUAG, CGAUAU, CGAUCA, CGAUCC, CGAUCG, CGAUCU, CGAUGA, CGAUGC, CGAUGG, CGAUGU, CGAUUA, CGAUUC, CGAUUG, CGAUUU, CGCAAA, CGCAAC, CGCAAG, CGCAAU, CGCACA, CGCACC, CGCACG, CGCAGA, CGCAGC, CGCAGG, CGCAGU, CGCAUA, CGCAUC, CGCAUG, CGCAUU, CGCCAA, CGCCAC, CGCCAG, CGCCAU, CGCCCA, CGCCCC, CGCCCG, CGCCGA, CGCCGC, CGCCGG, CGCCGU, CGCCUA, CGCCUG, CGCCUU, CGCGAA, CGCGAC, CGCGAG, CGCGAU, CGCGCA, CGCGCC, CGCGCG, CGCGCU, CGCGGA, CGCGGC, CGCGGG, CGCGGU, CGCGUA, CGCGUC, CGCGUG, CGCGUU, CGCUAA, CGCUAC, CGCUAG, CGCUAU, CGCUCA, CGCUCC, CGCUCG, CGCUCU, CGCUGA, CGCUGC, CGCUGG, CGCUGU, CGCUUA, CGCUUC, CGCUUG, CGGAAA, CGGAAC, CGGAAG, CGGACA, CGGACC, CGGACG, CGGACU, CGGAGC, CGGAGG, CGGAGU, CGGAUA, CGGAUU, CGGCAA, CGGCAC, CGGCAG, CGGCCA, CGGCCC, CGGCCG, CGGCGC, CGGCGG, CGGCGU, CGGCUA, CGGCUC, CGGCUG, CGGCUU, CGGGAA, CGGGAC, CGGGAG, CGGGAU, CGGGCA, CGGGCC, CGGGCG, CGGGCU, CGGGGU, CGGGUA, CGGGUC, CGGGUG, CGGUAA, CGGUAC, CGGUAG, CGGUAU, CGGUCA, CGGUCG, CGGUCU, CGGUGA, CGGUGG, CGGUGU, CGGUUA, CGGUUC, CGGUUG, CGGUUU, CGUAAA, CGUAAC, CGUAAG, CGUAAU, CGUACA, CGUACG, CGUACU, CGUAGA, CGUAGC, CGUAGG, CGUAGU, CGUAUA, CGUAUC, CGUAUG, CGUAUU, CGUCAA, CGUCAC, CGUCAG, CGUCAU, CGUCCA, CGUCCC, CGUCCG, CGUCCU, CGUCGA, CGUCGG, CGUCGU, CGUCUA, CGUCUC, CGUCUG, CGUCUU, CGUGAA, CGUGAC, CGUGAG, CGUGAU, CGUGCC, CGUGCG, CGUGCU, CGUGGA, CGUGGG, CGUGGU, CGUGUA, CGUGUG, CGUUAA, CGUUAC, CGUUAG, CGUUAU, CGUUCA, CGUUCC, CGUUCG, CGUUCU, CGUUGA, CGUUGC, CGUUGU, CGUUUA, CGUUUC, CGUUUU, CUAAAA, CUAAAC, CUAAAU, CUAACA, CUAACC, CUAACG, CUAACU, CUAAGA, CUAAGC, CUAAGU, CUAAUA, CUAAUC, CUAAUG, CUACAC, CUACAU, CUACCA, CUACCC, CUACCG, CUACCU, CUACGA, CUACGC, CUACGG, CUACGU, CUACUA, CUACUC, CUACUG, CUAGAA, CUAGAG, CUAGAU, CUAGCA, CUAGCC, CUAGCG, CUAGCU, CUAGGA, CUAGGG, CUAGGU, CUAGUG, CUAGUU, CUAUAA, CUAUAG, CUAUAU, CUAUCA, CUAUCC, CUAUCG, CUAUCU, CUAUGA, CUAUGC, CUAUGG, CUAUGU, CUAUUA, CUAUUG, CUCAAC, CUCAAG, CUCAAU, CUCACC, CUCACG, CUCAGC, CUCAUA, CUCAUC, CUCAUG, CUCAUU, CUCCAC, CUCCCC, CUCCCG, CUCCGA, CUCCGC, CUCCGG, CUCCUA, CUCCUC, CUCCUU, CUCGAA, CUCGAC, CUCGAG, CUCGAU, CUCGCA, CUCGCC, CUCGCG, CUCGGG, CUCGGU, CUCGUA, CUCGUC, CUCGUG, CUCGUU, CUCUAA, CUCUAC, CUCUAU, CUCUCA, CUCUCC, CUCUCU, CUCUGC, CUCUGU, CUCUUA, CUCUUG, CUGAAG, CUGACC, CUGACG, CUGAGC, CUGAUA, CUGAUC, CUGCCG, CUGCCU, CUGCGA, CUGCUA, CUGCUU, CUGGAG, CUGGAU, CUGGCG, CUGGGU, CUGUAC, CUGUCA, CUGUCC, CUGUCG, CUGUGG, CUGUGU, CUGUUA, CUGUUU, CUUAAC, CUUAAG, CUUAAU, CUUACC, CUUACG, CUUAGA, CUUAGC, CUUAGG, CUUAGU, CUUAUA, CUUAUC, CUUAUG, CUUAUU, CUUCAG, CUUCAU, CUUCCA, CUUCCC, CUUCCG, CUUCCU, CUUCGA, CUUCGC, CUUCGG, CUUCGU, CUUCUA, CUUGAC, CUUGAG, CUUGAU, CUUGCA, CUUGCC, CUUGCG, CUUGCU, CUUGGC, CUUGGU, CUUGUU, CUUUAC, CUUUAG, CUUUAU, CUUUCA, CUUUCG, CUUUCU, CUUUGA, CUUUGC, CUUUGU, CUUUUA, CUUUUC, CUUUUG, CUUUUU, GAAAAA, GAAAAG, GAAAAU, GAAACC, GAAACG, GAAAGA, GAAAGC, GAAAGU, GAAAUA, GAAAUC, GAAAUG, GAAAUU, GAACAA, GAACAC, GAACAG, GAACAU, GAACCA, GAACCC, GAACCG, GAACCU, GAACGA, GAACGC, GAACGG, GAACGU, GAACUA, GAACUG, GAACUU, GAAGAC, GAAGAG, GAAGCA, GAAGCG, GAAGCU, GAAGUC, GAAUAA, GAAUAC, GAAUAG, GAAUAU, GAAUCC, GAAUCG, GAAUCU, GAAUGA, GAAUGC, GAAUGU, GAAUUA, GAAUUC, GAAUUU, GACAAA, GACAAG, GACAAU, GACACC, GACAGA, GACAGG, GACAUA, GACAUG, GACAUU, GACCAA, GACCAC, GACCAG, GACCCA, GACCCC, GACCCG, GACCGC, GACCGG, GACCGU, GACCUA, GACCUC, GACCUU, GACGAA, GACGAC, GACGAG, GACGAU, GACGCA, GACGCC, GACGCG, GACGCU, GACGGA, GACGGC, GACGGG, GACGGU, GACGUA, GACGUC, GACGUG, GACGUU, GACUAA, GACUAC, GACUAG, GACUAU, GACUCA, GACUCC, GACUCG, GACUGG, GACUGU, GACUUA, GACUUG, GACUUU, GAGAAU, GAGAGA, GAGAGC, GAGAGG, GAGAUA, GAGAUC, GAGCAA, GAGCAU, GAGCCA, GAGCGA, GAGCGG, GAGCGU, GAGGGU, GAGGUC, GAGGUG, GAGUAA, GAGUAG, GAGUCC, GAGUUC, GAGUUU, GAUAAA, GAUAAC, GAUAAG, GAUAAU, GAUACA, GAUACC, GAUACG, GAUACU, GAUAGA, GAUAGC, GAUAGG, GAUAGU, GAUAUA, GAUCAA, GAUCAC, GAUCAU, GAUCCA, GAUCCC, GAUCCU, GAUCGC, GAUCGG, GAUCGU, GAUCUA, GAUCUG, GAUCUU, GAUGAA, GAUGAC, GAUGAG, GAUGCA, GAUGCC, GAUGCG, GAUGCU, GAUGGC, GAUGGG, GAUGGU, GAUGUG, GAUGUU, GAUUAA, GAUUAC, GAUUAG, GAUUAU, GAUUCA, GAUUCG, GAUUCU, GAUUGA, GAUUGC, GAUUUA, GAUUUC, GAUUUG, GAUUUU, GCAAAC, GCAAAG, GCAAAU, GCAACA, GCAACC, GCAAGC, GCAAGU, GCAAUA, GCAAUC, GCAAUG, GCAAUU, GCACAA, GCACAC, GCACAG, GCACCC, GCACCG, GCACCU, GCACGA, GCACGC, GCACGU, GCACUA, GCACUC, GCACUG, GCACUU, GCAGAU, GCAGCC, GCAGCG, GCAGGC, GCAGUA, GCAGUC, GCAGUG, GCAGUU, GCAUAA, GCAUAG, GCAUAU, GCAUCG, GCAUCU, GCAUGA, GCAUGC, GCAUGG, GCAUGU, GCAUUA, GCAUUC, GCAUUG, GCAUUU, GCCAAA, GCCAAC, GCCAAU, GCCACA, GCCACC, GCCACG, GCCAGA, GCCAGU, GCCAUA, GCCAUC, GCCAUG, GCCAUU, GCCCAA, GCCCAC, GCCCAG, GCCCCG, GCCCGA, GCCCGG, GCCCGU, GCCGAA, GCCGAC, GCCGAG, GCCGAU, GCCGCA, GCCGCU, GCCGGA, GCCGGC, GCCGGG, GCCGGU, GCCGUA, GCCGUC, GCCGUG, GCCGUU, GCCUAA, GCCUAU, GCCUCA, GCCUCC, GCCUCG, GCCUGA, GCCUUA, GCCUUU, GCGAAA, GCGAAC, GCGAAG, GCGAAU, GCGACC, GCGACG, GCGACU, GCGAGA, GCGAGC, GCGAGG, GCGAGU, GCGAUA, GCGAUC, GCGAUG, GCGAUU, GCGCAA, GCGCAC, GCGCAG, GCGCAU, GCGCCA, GCGCCC, GCGCCU, GCGCGA, GCGCGU, GCGCUA, GCGCUC, GCGCUG, GCGCUU, GCGGAA, GCGGAC, GCGGAU, GCGGCA, GCGGCC, GCGGCU, GCGGGA, GCGGUA, GCGGUC, GCGGUU, GCGUAA, GCGUAC, GCGUAG, GCGUAU, GCGUCA, GCGUCC, GCGUCG, GCGUCU, GCGUGA, GCGUGC, GCGUGG, GCGUGU, GCGUUA, GCGUUC, GCGUUG, GCGUUU, GCUAAA, GCUAAC, GCUAAG, GCUAAU, GCUACC, GCUACG, GCUACU, GCUAGA, GCUAGG, GCUAGU, GCUAUA, GCUAUC, GCUAUU, GCUCAA, GCUCAC, GCUCAG, GCUCAU, GCUCCA, GCUCCC, GCUCCG, GCUCGA, GCUCGC, GCUCGU, GCUCUA, GCUCUC, GCUCUU, GCUGAA, GCUGAC, GCUGAU, GCUGCA, GCUGCC, GCUGCG, GCUGCU, GCUGUG, GCUGUU, GCUUAC, GCUUAG, GCUUAU, GCUUCA, GCUUCG, GCUUGA, GCUUGG, GCUUGU, GCUUUA, GCUUUG, GGAAAG, GGAACA, GGAACC, GGAACG, GGAACU, GGAAGU, GGAAUA, GGAAUC, GGAAUU, GGACAA, GGACAC, GGACAG, GGACAU, GGACCG, GGACGA, GGACGC, GGACGU, GGACUA, GGACUC, GGACUU, GGAGAC, GGAGCA, GGAGCG, GGAGGG, GGAGUA, GGAUAA, GGAUAC, GGAUCA, GGAUCC, GGAUCG, GGAUCU, GGAUGC, GGAUUA, GGAUUG, GGCAAU, GGCACA, GGCACU, GGCAGA, GGCAUA, GGCAUC, GGCCAC, GGCCAG, GGCCCC, GGCCGA, GGCCGC, GGCCGU, GGCCUA, GGCCUG, GGCCUU, GGCGAA, GGCGAG, GGCGAU, GGCGCA, GGCGCU, GGCGGU, GGCGUA, GGCGUC, GGCGUG, GGCGUU, GGCUAA, GGCUAC, GGCUAG, GGCUAU, GGCUCC, GGCUCG, GGCUGA, GGCUUA, GGCUUC, GGCUUG, GGGAAU, GGGACA, GGGAGA, GGGAGU, GGGAUA, GGGAUU, GGGCAA, GGGCAC, GGGCAG, GGGCCG, GGGCGG, GGGGCC, GGGGGG, GGGGGU, GGGGUA, GGGUAC, GGGUAU, GGGUCA, GGGUCC, GGGUCG, GGGUGA, GGGUGC, GGGUUA, GGGUUG, GGUAAA, GGUAAC, GGUAAG, GGUAAU, GGUACA, GGUACC, GGUACG, GGUACU, GGUAGC, GGUAGG, GGUAGU, GGUAUA, GGUAUC, GGUAUG, GGUCAA, GGUCAC, GGUCAG, GGUCAU, GGUCCA, GGUCCG, GGUCCU, GGUCGA, GGUCGC, GGUCGG, GGUCGU, GGUCUC, GGUCUU, GGUGAA, GGUGAC, GGUGAU, GGUGCA, GGUGCC, GGUGGC, GGUGUA, GGUGUC, GGUUAA, GGUUAG, GGUUAU, GGUUCA, GGUUCC, GGUUCG, GGUUGC, GGUUUC, GGUUUU, GUAAAA, GUAAAG, GUAAAU, GUAACC, GUAACG, GUAACU, GUAAGA, GUAAGC, GUAAGG, GUAAGU, GUAAUA, GUAAUC, GUAAUG, GUAAUU, GUACAA, GUACAC, GUACAG, GUACAU, GUACCA, GUACCC, GUACCG, GUACCU, GUACGA, GUACGC, GUACGG, GUACGU, GUACUA, GUACUC, GUACUG, GUACUU, GUAGAA, GUAGAC, GUAGCA, GUAGCC, GUAGCG, GUAGCU, GUAGGA, GUAGGC, GUAGGG, GUAGGU, GUAGUA, GUAGUC, GUAUAA, GUAUAC, GUAUAG, GUAUAU, GUAUCA, GUAUCG, GUAUCU, GUAUGA, GUAUGC, GUAUGG, GUAUUA, GUAUUG, GUAUUU, GUCAAA, GUCAAG, GUCAAU, GUCACA, GUCACC, GUCACG, GUCAGA, GUCAGC, GUCAGG, GUCAUA, GUCAUC, GUCAUG, GUCCAA, GUCCAC, GUCCAU, GUCCCC, GUCCCU, GUCCGA, GUCCGC, GUCCGG, GUCCGU, GUCCUA, GUCCUG, GUCCUU, GUCGAA, GUCGAC, GUCGAG, GUCGAU, GUCGCA, GUCGCC, GUCGCG, GUCGCU, GUCGGA, GUCGGC, GUCGGG, GUCGGU, GUCGUA, GUCGUC, GUCGUU, GUCUAA, GUCUAG, GUCUCA, GUCUCC, GUCUCG, GUCUGA, GUCUGG, GUCUGU, GUCUUC, GUCUUU, GUGAAA, GUGAAC, GUGAAG, GUGACC, GUGACG, GUGAGA, GUGAGC, GUGAGU, GUGAUC, GUGAUG, GUGAUU, GUGCAC, GUGCAU, GUGCCC, GUGCCG, GUGCGA, GUGCGG, GUGCGU, GUGCUA, GUGCUC, GUGCUG, GUGGAG, GUGGCG, GUGGCU, GUGGGU, GUGGUC, GUGGUG, GUGUAA, GUGUAG, GUGUCG, GUGUGA, GUGUGC, GUGUGU, GUGUUG, GUGUUU, GUUAAA, GUUAAC, GUUAAG, GUUACA, GUUACC, GUUACG, GUUACU, GUUAGA, GUUAGC, GUUAGU, GUUAUA, GUUAUC, GUUAUG, GUUAUU, GUUCAA, GUUCAC, GUUCAG, GUUCCA, GUUCCG, GUUCGA, GUUCGC, GUUCGG, GUUCGU, GUUCUA, GUUCUG, GUUGAA, GUUGAC, GUUGAG, GUUGAU, GUUGCG, GUUGCU, GUUGGA, GUUGGC, GUUGGU, GUUGUC, GUUGUG, GUUGUU, GUUUAA, GUUUAC, GUUUAG, GUUUAU, GUUUCA, GUUUCC, GUUUCU, GUUUGA, GUUUGC, GUUUGG, GUUUGU, GUUUUA, GUUUUC, GUUUUU, UAAAAA, UAAAAC, UAAAAG, UAAAAU, UAAACA, UAAACC, UAAACG, UAAACU, UAAAGA, UAAAGG, UAAAGU, UAAAUA, UAAAUC, UAAAUG, UAAAUU, UAACAA, UAACAC, UAACAG, UAACCA, UAACCC, UAACCG, UAACCU, UAACGA, UAACGC, UAACGG, UAACGU, UAACUA, UAACUG, UAACUU, UAAGAG, UAAGAU, UAAGCA, UAAGCC, UAAGCG, UAAGCU, UAAGGA, UAAGGC, UAAGGG, UAAGGU, UAAGUA, UAAGUC, UAAGUG, UAAGUU, UAAUAA, UAAUCA, UAAUCC, UAAUCG, UAAUCU, UAAUGA, UAAUGG, UAAUGU, UAAUUA, UAAUUC, UAAUUG, UACAAC, UACAAG, UACAAU, UACACC, UACACG, UACACU, UACAGA, UACAGC, UACAUA, UACAUC, UACAUU, UACCAA, UACCAC, UACCAG, UACCAU, UACCCC, UACCCG, UACCCU, UACCGA, UACCGC, UACCGG, UACCGU, UACCUA, UACCUG, UACGAA, UACGAC, UACGAG, UACGAU, UACGCA, UACGCC, UACGCG, UACGCU, UACGGC, UACGGG, UACGGU, UACGUA, UACGUC, UACGUG, UACGUU, UACUAA, UACUAC, UACUAG, UACUAU, UACUCA, UACUCC, UACUCG, UACUCU, UACUGA, UACUGC, UACUGG, UACUUA, UACUUG, UACUUU, UAGAAA, UAGAAG, UAGAAU, UAGACA, UAGACG, UAGAGA, UAGAGC, UAGAGU, UAGAUA, UAGAUC, UAGAUG, UAGCAU, UAGCCC, UAGCCG, UAGCCU, UAGCGA, UAGCGC, UAGCGU, UAGCUA, UAGCUC, UAGCUG, UAGGAA, UAGGAU, UAGGCG, UAGGCU, UAGGGU, UAGGUC, UAGGUG, UAGGUU, UAGUAA, UAGUAC, UAGUAG, UAGUAU, UAGUCA, UAGUCG, UAGUGU, UAGUUA, UAGUUC, UAGUUG, UAGUUU, UAUAAC, UAUAAG, UAUACU, UAUAGA, UAUAGC, UAUAGG, UAUAGU, UAUAUA, UAUAUC, UAUAUG, UAUAUU, UAUCAA, UAUCAC, UAUCAU, UAUCCA, UAUCCC, UAUCCG, UAUCCU, UAUCGA, UAUCGC, UAUCGG, UAUCGU, UAUCUA, UAUCUC, UAUCUG, UAUCUU, UAUGAA, UAUGAC, UAUGAG, UAUGAU, UAUGCA, UAUGCG, UAUGCU, UAUGGA, UAUGGC, UAUGUC, UAUGUG, UAUGUU, UAUUAG, UAUUCA, UAUUCC, UAUUCG, UAUUCU, UAUUGA, UAUUGG, UAUUUA, UAUUUC, UAUUUG, UAUUUU, UCAAAA, UCAAAC, UCAAAG, UCAACC, UCAACU, UCAAGA, UCAAGC, UCAAUA, UCAAUC, UCAAUG, UCAAUU, UCACCC, UCACCG, UCACCU, UCACGA, UCACGC, UCACGG, UCACGU, UCACUA, UCACUC, UCACUU, UCAGAA, UCAGAC, UCAGAG, UCAGCG, UCAGCU, UCAGGA, UCAGGC, UCAGGU, UCAGUC, UCAGUU, UCAUAA, UCAUCA, UCAUCC, UCAUCG, UCAUGC, UCAUGG, UCAUGU, UCAUUA, UCAUUG, UCCAAA, UCCAAC, UCCAAG, UCCAAU, UCCACA, UCCACC, UCCACG, UCCAGC, UCCAGG, UCCAUA, UCCAUC, UCCAUU, UCCCAA, UCCCAG, UCCCAU, UCCCCC, UCCCCG, UCCCCU, UCCCGA, UCCCGC, UCCCGG, UCCCGU, UCCCUA, UCCCUC, UCCGAA, UCCGAC, UCCGAG, UCCGAU, UCCGCA, UCCGCC, UCCGGA, UCCGGC, UCCGGU, UCCGUA, UCCGUC, UCCGUG, UCCUAA, UCCUCA, UCCUCG, UCCUCU, UCCUGC, UCCUGU, UCCUUA, UCCUUC, UCCUUU, UCGAAA, UCGAAC, UCGAAG, UCGAAU, UCGACA, UCGACC, UCGACG, UCGACU, UCGAGA, UCGAGC, UCGAGG, UCGAUA, UCGAUC, UCGAUG, UCGAUU, UCGCAA, UCGCAC, UCGCAG, UCGCAU, UCGCCA, UCGCCC, UCGCCG, UCGCCU, UCGCGA, UCGCGC, UCGCGU, UCGCUA, UCGCUC, UCGGAA, UCGGAC, UCGGAG, UCGGAU, UCGGCA, UCGGCU, UCGGGG, UCGGGU, UCGGUC, UCGGUG, UCGGUU, UCGUAA, UCGUAC, UCGUAG, UCGUAU, UCGUCA, UCGUCC, UCGUCG, UCGUCU, UCGUGA, UCGUGU, UCGUUA, UCGUUC, UCGUUG, UCGUUU, UCUAAC, UCUAAG, UCUAAU, UCUACA, UCUACC, UCUACG, UCUACU, UCUAGC, UCUAGG, UCUAGU, UCUAUA, UCUAUC, UCUAUG, UCUAUU, UCUCAG, UCUCAU, UCUCCG, UCUCGC, UCUCGG, UCUCGU, UCUCUC, UCUGAA, UCUGAU, UCUGCA, UCUGCG, UCUGCU, UCUGGC, UCUGGU, UCUGUC, UCUGUG, UCUGUU, UCUUAA, UCUUAC, UCUUAG, UCUUAU, UCUUCA, UCUUCC, UCUUCG, UCUUCU, UCUUGC, UCUUGG, UCUUGU, UCUUUA, UCUUUC, UCUUUG, UCUUUU, UGAAAA, UGAAAC, UGAACA, UGAACC, UGAAGG, UGAAUC, UGAAUG, UGACAA, UGACAC, UGACAG, UGACCA, UGACCC, UGACCG, UGACGA, UGACGC, UGACGG, UGACGU, UGACUA, UGACUC, UGACUU, UGAGAG, UGAGAU, UGAGCA, UGAGCC, UGAGCU, UGAGGC, UGAGGU, UGAGUA, UGAGUU, UGAUAC, UGAUAG, UGAUAU, UGAUCA, UGAUCG, UGAUCU, UGAUGA, UGAUGC, UGAUGG, UGAUGU, UGAUUA, UGAUUC, UGAUUG, UGAUUU, UGCAAC, UGCAAG, UGCACA, UGCACG, UGCAGG, UGCAGU, UGCAUC, UGCCCA, UGCCCC, UGCCCG, UGCCGA, UGCCGC, UGCCGG, UGCCGU, UGCCUA, UGCCUC, UGCCUG, UGCCUU, UGCGAA, UGCGAC, UGCGAU, UGCGCC, UGCGCG, UGCGCU, UGCGGC, UGCGGG, UGCGGU, UGCGUA, UGCGUC, UGCGUG, UGCGUU, UGCUAC, UGCUAU, UGCUCC, UGCUCG, UGCUGC, UGCUGG, UGCUGU, UGCUUA, UGCUUU, UGGAAC, UGGAAG, UGGAGC, UGGAUC, UGGAUU, UGGCAA, UGGCAC, UGGCAG, UGGCCG, UGGCCU, UGGCGA, UGGCGC, UGGCGU, UGGCUA, UGGCUC, UGGCUU, UGGGAA, UGGGCA, UGGGCC, UGGGGC, UGGGUC, UGGUAA, UGGUAG, UGGUAU, UGGUCC, UGGUCG, UGGUCU, UGGUGA, UGGUGC, UGGUGG, UGGUGU, UGGUUA, UGGUUG, UGUAAA, UGUAAC, UGUAAG, UGUACC, UGUACG, UGUACU, UGUAGA, UGUAGC, UGUAGU, UGUAUC, UGUAUU, UGUCAA, UGUCAC, UGUCAG, UGUCAU, UGUCCA, UGUCCC, UGUCCG, UGUCGA, UGUCGC, UGUCGG, UGUCGU, UGUCUA, UGUCUC, UGUGAC, UGUGAG, UGUGAU, UGUGCA, UGUGGU, UGUGUA, UGUGUU, UGUUAC, UGUUAG, UGUUAU, UGUUCA, UGUUCC, UGUUCG, UGUUGG, UGUUGU, UGUUUA, UGUUUC, UGUUUG, UGUUUU, UUAAAA, UUAAAC, UUAAAG, UUAAAU, UUAACC, UUAACG, UUAACU, UUAAGU, UUAAUA, UUAAUC, UUAAUG, UUAAUU, UUACAA, UUACAC, UUACAG, UUACAU, UUACCA, UUACCC, UUACCG, UUACCU, UUACGA, UUACGC, UUACGG, UUACGU, UUACUA, UUACUC, UUACUG, UUACUU, UUAGAA, UUAGAC, UUAGCC, UUAGCG, UUAGCU, UUAGGC, UUAGGU, UUAGUA, UUAGUC, UUAGUU, UUAUAA, UUAUAC, UUAUAG, UUAUAU, UUAUCC, UUAUCG, UUAUCU, UUAUGA, UUAUGG, UUAUGU, UUAUUA, UUAUUC, UUAUUG, UUAUUU, UUCAAC, UUCAAU, UUCACA, UUCACC, UUCACG, UUCACU, UUCAGC, UUCAGG, UUCAGU, UUCAUA, UUCAUC, UUCAUG, UUCAUU, UUCCAA, UUCCCA, UUCCCG, UUCCGA, UUCCGU, UUCCUU, UUCGAA, UUCGAC, UUCGAG, UUCGAU, UUCGCA, UUCGCC, UUCGCG, UUCGCU, UUCGGA, UUCGGC, UUCGGG, UUCGGU, UUCGUA, UUCGUC, UUCGUG, UUCGUU, UUCUAC, UUCUAG, UUCUCA, UUCUCG, UUCUGG, UUCUUA, UUCUUU, UUGAAA, UUGAAC, UUGAAG, UUGAAU, UUGACC, UUGACG, UUGACU, UUGAGA, UUGAGC, UUGAGU, UUGAUA, UUGAUC, UUGAUG, UUGAUU, UUGCAA, UUGCAC, UUGCAG, UUGCAU, UUGCCC, UUGCCG, UUGCGA, UUGCGC, UUGCGG, UUGCGU, UUGCUA, UUGCUC, UUGCUG, UUGCUU, UUGGAA, UUGGAG, UUGGCC, UUGGCG, UUGGCU, UUGGGC, UUGGGU, UUGGUA, UUGGUG, UUGUAA, UUGUAC, UUGUCA, UUGUCG, UUGUCU, UUGUGC, UUGUGG, UUGUUA, UUGUUG, UUGUUU, UUUAAA, UUUAAC, UUUAAG, UUUAAU, UUUACA, UUUACC, UUUACG, UUUACU, UUUAGA, UUUAGC, UUUAGG, UUUAGU, UUUAUA, UUUAUC, UUUAUG, UUUAUU, UUUCAU, UUUCCA, UUUCCG, UUUCCU, UUUCGA, UUUCGC, UUUCGG, UUUCGU, UUUCUA, UUUCUC, UUUCUG, UUUCUU, UUUGAA, UUUGAC, UUUGAG, UUUGAU, UUUGCC, UUUGCU, UUUGGA, UUUGGC, UUUGGG, UUUGGU, UUUGUA, UUUGUC, UUUGUU, UUUUAA, UUUUAG, UUUUAU, UUUUCC, UUUUCG, UUUUCU, UUUUGA, UUUUGC, UUUUGG, UUUUGU, UUUUUA, UUUUUC,  UUUUUU

TABLE 2 Oligonucleotide sequences made for testing in the lab. Gene ExptCell Assay Oligo ID RQ RQ SE Name Type Line/Tissue [Oligo] Type BDNF-010.658347457 0 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-01 0.2825133540.080648333 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-01 2.0206915741.773367664 BDNF in vivo liv mus C57BI 25 qRTPCR m02 BDNF-01 1.8905907270.480550547 BDNF in vivo liv mus C57BI 10 qRTPCR m02 BDNF-02 1.5072524790 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-02 0.181361249 0.016360562 BDNFin vitro Hep3B 10 qRTPCR m02 BDNF-02 4.682822957 2.116973542 BDNF invivo liv mus C57BI 25 qRTPCR m02 BDNF-02 1.397329188 0.449770036 BDNF invivo liv mus C57BI 10 qRTPCR m02 BDNF-03 0.433941497 0.112936272 BDNF invitro Hep3B 30 qRTPCR m02 BDNF-03 0.704472154 0.107898872 BDNF in vitroHep3B 10 qRTPCR m02 BDNF-03 1.02120249 0.288262867 BDNF in vivo kid mus25 qRTPCR m02 C57BI BDNF-03 0.834019396 0.034886258 BDNF in vivo kid mus10 qRTPCR m02 C57BI BDNF-03 3.239625501 1.535293608 BDNF in vivo liv musC57BI 25 qRTPCR m02 BDNF-03 2.002007629 21.58110056 BDNF in vivo liv musC57BI 10 qRTPCR m02 BDNF-04 1.2391966 0.380159297 BDNF in vitro Hep3B 30qRTPCR m02 BDNF-04 1.836853076 0.169112065 BDNF in vitro Hep3B 10 qRTPCRm02 BDNF-04 0.896518494 0.137801624 BDNF in vivo kid mus 25 qRTPCR m02C57BI BDNF-04 0.892584372 0.111930079 BDNF in vivo kid mus 10 qRTPCR m02C57BI BDNF-04 100.1014559 64.41364711 BDNF in vivo liv mus C57BI 25qRTPCR m02 BDNF-04 186.0293662 160.6875261 BDNF in vivo liv mus C57BI 10qRTPCR m02 BDNF-05 0.737305708 0.230845985 BDNF in vitro Hep3B 30 qRTPCRm02 BDNF-05 0.976448173 0.178758021 BDNF in vitro Hep3B 10 qRTPCR m02BDNF-05 2.226854577 1.000703266 BDNF in vivo kid mus 25 qRTPCR m02 C57BIBDNF-05 1.753619037 0.288958367 BDNF in vivo kid mus 10 qRTPCR m02 C57BIBDNF-05 6.161706234 1.548844787 BDNF in vivo liv mus C57BI 25 qRTPCR m02BDNF-05 17.60936455 11.73293839 BDNF in vivo liv mus C57BI 10 qRTPCR m02BDNF-06 0.245648671 0.113950319 BDNF in vitro Hep3B 30 qRTPCR m02BDNF-06 0.639126937 0.088822258 BDNF in vitro Hep3B 10 qRTPCR m02BDNF-06 1.604050297 0.192586674 BDNF in vivo kid mus 25 qRTPCR m02 C57BIBDNF-06 0.584668058 0.039328809 BDNF in vivo kid mus 10 qRTPCR m02 C57BIBDNF-06 27.40525087 12.16497094 BDNF in vivo liv mus C57BI 25 qRTPCR m02BDNF-06 37.63954563 42.48273569 BDNF in vivo liv mus C57BI 10 qRTPCR m02BDNF-07 0.74168684 0 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-071.472499536 0.281748996 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-070.45376548 0.036386394 BDNF in vivo kid mus 25 qRTPCR m02 C57BI BDNF-070.390421554 0.026369826 BDNF in vivo kid mus 10 qRTPCR m02 C57BI BDNF-0711.28088892 6.55145925 BDNF in vivo liv mus C57BI 25 qRTPCR m02 BDNF-078.78102149 7.061030116 BDNF in vivo liv mus C57BI 10 qRTPCR m02 BDNF-080.718642912 0.052875505 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-081.192721717 0.315986688 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-090.616717484 0.1250181 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-091.352856657 0.435426216 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-102.316633163 0.761276801 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-101.650321252 0.449925321 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-111.017920155 0.186151377 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-111.42872483 0.330772184 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-110.938061845 0.052194407 BDNF in vivo kid mus 25 qRTPCR m02 C57BI BDNF-111.015189977 0.199591111 BDNF in vivo kid mus 10 qRTPCR m02 C57BI BDNF-120.543203889 0.085552972 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-120.624397993 0.033331468 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-130.311216669 0.114540021 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-131.343782534 0.132030095 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-141.291728767 0.220756514 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-142.045726366 0.196078413 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-151.260802698 0.168013451 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-151.578814681 0.242398686 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-169.483353327 0.962953672 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-163.385954439 0.479571813 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-176.254734831 0.75286845 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-173.778966926 0.725404307 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-180.831110344 0.084627511 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-181.437628645 0.225973805 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-181.262070396 0.229953382 BDNF in vivo kid mus 25 qRTPCR m02 C57BI BDNF-180.960812942 0.043540533 BDNF in vivo kid mus 10 qRTPCR m02 C57BI BDNF-190.50238621 0.123477791 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-191.721571854 0.393106998 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-200.678955385 0.061672891 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-201.363651775 0.291599508 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-210.38968081 0.072631587 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-210.762849412 0.180532241 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-220.480690649 0.147239665 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-220.617499352 0.263150195 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-230.665875976 0.457524278 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-230.644627324 0.160313284 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-240.19580671 0.082872733 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-241.158636252 0.206418695 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-253.225781118 0.172512002 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-254.395555504 0.442543763 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-261.356992496 NA BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-26 1.5451175050.376307394 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-27 1.0451368020.169520404 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-27 1.8444420210.26755814 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-28 0.6689970220.1570761 BDNF in vitro Hep3B 30 qRTPCR m02 BDNF-28 1.1306394170.120737074 BDNF in vitro Hep3B 10 qRTPCR m02 BDNF-29 1.099556016 NABDNF in vitro Hep3B 30 qRTPCR m02 BDNF-29 0.939912499 0.155918621 BDNFin vitro Hep3B 10 qRTPCR m02 BDNF-30 0.8073181 NA BDNF in vitro Hep3B 30qRTPCR m02 BDNF-30 1.632544933 0.253640398 BDNF in vitro Hep3B 10 qRTPCRm02

TABLE 4 Formatted oligonucleotide sequences made fortesting in the lab showing nucleotide modifications. OligoIDBase Sequence Formatted Sequence SeqID BDNF-01 m02 AAGTATGAAATAACCdAs;InaAs;dGs;InaTs;dAs;InaTs;dGs;InaAs;dAs; 42269InaAs;dTs;InaAs;dAs;InaCs;dC-Sup BDNF-02 m02 AGTAAGGAAAAGGATdAs;InaGs;dTs;InaAs;dAs;InaGs;dGs;InaAs;dAs; 42286InaAs;dAs;InaGs;dGs;InaAs;dT-Sup BDNF-03 m02 TCTCACCTGGTGGAAdTs;InaCs;dTs;InaCs;dAs;InaCs;dCs;InaTs;dGs; 42313InaGs;dTs;InaGs;dGs;InaAs;dA-Sup BDNF-04 m02 AGAAAGCAGAAACAAdAs;InaGs;dAs;InaAs;dAs;InaGs;dCs;InaAs;dGs; 42336InaAs;dAs;InaAs;dCs;InaAs;dA-Sup BDNF-05 m02 AAACAAGACAGAAAAdAs;InaAs;dAs;InaCs;dAs;InaAs;dGs;InaAs;dCs; 42345InaAs;dGs;InaAs;dAs;InaAs;dA-Sup BDNF-06 m02 ATGTTGCTAACTTGAdAs;InaTs;dGs;InaTs;dTs;InaGs;dCs;InaTs;dAs; 8673InaAs;dCs;InaTs;dTs;InaGs;dA-Sup BDNF-07 m02 ACACTTGCAGTTGTTdAs;InaCs;dAs;InaCs;dTs;InaTs;dGs;InaCs;dAs; 8640InaGs;dTs;InaTs;dGs;InaTs;dT-Sup BDNF-08 m02 TGTTTCTCGTGACAGdTs;InaGs;dTs;InaTs;dTs;InaCs;dTs;InaCs;dGs; 8602InaTs;dGs;InaAs;dCs;InaAs;dG-Sup BDNF-09 m02 AGGCGAGGAGGAGGAdAs;InaGs;dGs;InaCs;dGs;InaAs;dGs;InaGs;dAs; 62210InaGs;dGs;InaAs;dGs;InaGs;dA-Sup BDNF-10 m02 AGAGAACGAAGCTAGdAs;InaGs;dAs;InaGs;dAs;InaAs;dCs;InaGs;dAs; 62211InaAs;dGs;InaCs;dTs;InaAs;dG-Sup BDNF-11 m02 ACCAGAATCAAAATTdAs;InaCs;dCs;InaAs;dGs;InaAs;dAs;InaTs;dCs; 54711InaAs;dAs;InaAs;dAs;InaTs;dT-Sup BDNF-12 m02 TATTAGAAGAGTTCCdTs;InaAs;dTs;InaTs;dAs;InaGs;dAs;InaAs;dGs; 54752InaAs;dGs;InaTs;dTs;InaCs;dC-Sup BDNF-13 m02 AGGGCATTGCATGCTdAs;InaGs;dGs;InaGs;dCs;InaAs;dTs;InaTs;dGs; 54772InaCs;dAs;InaTs;dGs;InaCs;dT-Sup BDNF-14 m02 CTCGCTCATTCATTAdCs;InaTs;dCs;InaGs;dCs;InaTs;dCs;InaAs;dTs; 28776InaTs;dCs;InaAs;dTs;InaTs;dA-Sup BDNF-15 m02 AAGAACTCAAAAGGAdAs;InaAs;dGs;InaAs;dAs;InaCs;dTs;InaCs;dAs; 28752InaAs;dAs;InaAs;dGs;InaGs;dA-Sup BDNF-16 m02 AAACGTGTCTCTCGGdAs;InaAs;dAs;InaCs;dGs;InaTs;dGs;InaTs;dCs; 28738InaTs;dCs;InaTs;dCs;InaGs;dG-Sup BDNF-17 m02 TCAGAGCGACGGACAdTs;InaCs;dAs;InaGs;dAs;InaGs;dCs;InaGs;dAs; 62212InaCs;dGs;InaGs;dAs;InaCs;dA-Sup BDNF-18 m02 ACAACTGGTCTACATdAs;InaCs;dAs;InaAs;dCs;InaTs;dGs;InaGs;dTs; 62213InaCs;dTs;InaAs;dCs;InaAs;dT-Sup BDNF-19 m02 TTCTGCTCTGCTGTGdTs;InaTs;dCs;InaTs;dGs;InaCs;dTs;InaCs;dTs; 62214InaGs;dCs;InaTs;dGs;InaTs;dG-Sup BDNF-20 m02 TGAAACTTAAGAGCTdTs;InaGs;dAs;InaAs;dAs;InaCs;dTs;InaTs;dAs; 62215InaAs;dGs;InaAs;dGs;InaCs;dT-Sup BDNF-21 m02 CCACGGCCAGGGCCTdCs;InaCs;dAs;InaCs;dGs;InaGs;dCs;InaCs;dAs; 62216InaGs;dGs;InaGs;dCs;InaCs;dT-Sup BDNF-22 m02 ACAGTGATCTGCACAdAs;InaCs;dAs;InaGs;dTs;InaGs;dAs;InaTs;dCs; 62217InaTs;dGs;InaCs;dAs;InaCs;dA-Sup BDNF-23 m02 ATCGCATTAGTGCCCdAs;InaTs;dCs;InaGs;dCs;InaAs;dTs;InaTs;dAs; 62218InaGs;dTs;InaGs;dCs;InaCs;dC-Sup BDNF-24 m02 TGTATACCCCATCTTdTs;InaGs;dTs;InaAs;dTs;InaAs;dCs;InaCs;dCs; 62219InaCs;dAs;InaTs;dCs;InaTs;dT-Sup BDNF-25 m02 AATCAGCCCTCATTTdAs;InaAs;dTs;InaCs;dAs;InaGs;dCs;InaCs;dCs; 62220InaTs;dCs;InaAs;dTs;InaTs;dT-Sup BDNF-26 m02 TTAGGCGAAGCCCCAdTs;InaTs;dAs;InaGs;dGs;InaCs;dGs;InaAs;dAs; 62221InaGs;dCs;InaCs;dCs;InaCs;dA-Sup BDNF-27 m02 TCTAAATGGCCCATGdTs;InaCs;dTs;InaAs;dAs;InaAs;dTs;InaGs;dGs; 62222InaCs;dCs;InaCs;dAs;InaTs;dG-Sup BDNF-28 m02 TATCTCCAAGCCCTTdTs;InaAs;dTs;InaCs;dTs;InaCs;dCs;InaAs;dAs; 62223InaGs;dCs;InaCs;dCs;InaTs;dT-Sup BDNF-29 m02 ATCTCCTACAGAGATdAs;InaTs;dCs;InaTs;dCs;InaCs;dTs;InaAs;dCs; 62224InaAs;dGs;InaAs;dGs;InaAs;dT-Sup BDNF-30 m02 ACTGGTGTACCCAATdAs;InaCs;dTs;InaGs;dGs;InaTs;dGs;InaTs;dAs; 62225InaCs;dCs;InaCs;dAs;InaAs;dT-Sup

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID Chrom Gene Chr. Start Chr. End Strand 1 chr11 BDNF 2766444127693196 − 2 chr11 BDNF 27664441 27693196 + 3 chr11 BDNF-AS1 2751639827731718 + 4 chr11 BDNF-AS1 27516398 27731718 − 5 chr2 Bdnf 109502856109579200 + 6 chr2 Bdnf 109502856 109579200 − 7 chr11 BDNF 2767881927678888 − 8 chr11 BDNF 27679423 27679469 − 9 chr11 BDNF 2767951227679558 − 10 chr11 BDNF 27679705 27679749 − 11 chr11 BDNF 2768665727686742 − 12 chr11 BDNF 27718502 27718548 − 13 chr11 BDNF 2771974327719780 − 14 chr11 BDNF 27721391 27721434 − 15 chr11 BDNF 2767681927680888 − 16 chr11 BDNF 27677423 27681469 − 17 chr11 BDNF 2767751227681558 − 18 chr11 BDNF 27677705 27681749 − 19 chr11 BDNF 2768465727688742 − 20 chr11 BDNF 27716502 27720548 − 21 chr11 BDNF 2771774327721780 − 22 chr11 BDNF 27719391 27723434 − 23 chr11 BDNF 2773923027739276 − 24 chr11 BDNF 27741576 27741622 − 25 chr11 BDNF 2774248127742526 − 26 chr11 BDNF 27742552 27742602 − 27 chr11 BDNF 2773723027741276 − 28 chr11 BDNF 27739576 27743622 − 29 chr11 BDNF 2774048127744526 − 30 chr11 BDNF 27740552 27744602 − 31 chr11 BDNF 2751852727518574 − 32 chr11 BDNF 27518780 27518823 − 33 chr11 BDNF 2751887027518922 − 34 chr11 BDNF 27519285 27519333 − 35 chr11 BDNF 2752049827520547 − 36 chr11 BDNF 27520913 27520996 − 37 chr11 BDNF 2752108127521112 − 38 chr11 BDNF 27523348 27523395 − 39 chr11 BDNF 2752342327523469 − 40 chr11 BDNF 27516527 27520574 − 41 chr11 BDNF 2751678027520823 − 42 chr11 BDNF 27516870 27520922 − 43 chr11 BDNF 2751728527521333 − 44 chr11 BDNF 27518498 27522547 − 45 chr11 BDNF 2751891327522996 − 46 chr11 BDNF 27519081 27523112 − 47 chr11 BDNF 2752134827525395 − 48 chr11 BDNF 27521423 27525469 − 49 chr11 BDNF 2768191727681964 + 50 chr11 BDNF 27697978 27698023 + 51 chr11 BDNF 2771859927718680 + 52 chr11 BDNF 27679917 27683964 + 53 chr11 BDNF 2769597827700023 + 54 chr11 BDNF 27716599 27720680 + 55 chr11 BDNF 2752370827523784 + 56 chr11 BDNF 27527959 27528009 + 57 chr11 BDNF 2752806327528106 + 58 chr11 BDNF 27521708 27525784 + 59 chr11 BDNF 2752595927530009 + 60 chr11 BDNF 27526063 27530106 + 61 chr11 BDNF 2773483627734884 + 62 chr11 BDNF 27740311 27740344 + 63 chr11 BDNF 2774178627741828 + 64 chr11 BDNF 27742450 27742493 + 65 chr11 BDNF 2773283627736884 + 66 chr11 BDNF 27738311 27742344 + 67 chr11 BDNF 2773978627743828 + 68 chr11 BDNF 27740450 27744493 +

Single Strand Oligonucleotides (Antisense Strand of Target Gene BDNF)

SegID range: 69-30107, 62210-62225

SegIDs w/o G Runs:

69-119, 140-142, 156-165, 179-227, 241-380, 395-559, 573-588, 602-849,876-967, 982-1636, 1650-2761, 2765-2817, 2832-2869, 2877-2902,2916-3015, 3029-3199, 3214-3216, 3230-3409, 3418-3447, 3461-3524,3538-3568, 3595-3641, 3655-3721, 3735-3757, 3771-3822, 3824-3920,3934-4107, 4121-4257, 4274-4433, 4447-4733, 4747-4749, 4763-4905,4919-4978, 4992-5624, 5638-5699, 5719-6026, 6040-6090, 6104-6121,6136-6142, 6171-6209, 6224-6230, 6244-6245, 6259-6437, 6453-6484,6498-6499, 6520-6521, 6535-6561, 6584-6607, 6621-6778, 6792-7030,7044-7117, 7142-7199, 7214-7294, 7308-7573, 7586-7622, 7636-7692,7717-7742, 7756, 7770-7804, 7819-7981, 7995-8083, 8097-8104, 8118-8316,8330-8459, 8475-8540, 8554-8826, 8840-8961, 8975-9166, 9180-9208,9222-9419, 9433-9565, 9579-9646, 9672-9769, 9783-9919, 9933-10186,10200-10206, 10220-10461, 10475-10740, 10754-10813, 10841-10967,10981-10985, 10999-11105, 11119, 11133-11143, 11157-11174, 11188-11239,11253-11341, 11361-11477, 11491-11544, 11559-11583, 11597-11622,11636-11652, 11666-11671, 11683-11736, 11761-11861, 11877-11981,12002-12047, 12061-12072, 12086-12270, 12284-12552, 12566-12573,12587-12588, 12602-12716, 12730-12751, 12766-12815, 12829-12953,12967-13131, 13156-13167, 13181-13189, 13203-13449, 13471-13487,13501-13535, 13549-13764, 13778-13855, 13883-13923, 13935-14121,14136-14296, 14310-14332, 14346-14591, 14605-14668, 14682-14803,14817-14861, 14875-15470, 15484-15519, 15533-15565, 15579-15585,15599-15660, 15674-15705, 15719-15779, 15793-15812, 15826-15855,15869-16013, 16035-16195, 16209-16216, 16230-16372, 16386-16680,16707-16713, 16727-16815, 16829-16951, 16977-17665, 17679-17707,17722-18003, 18017-18056, 18072-18110, 18125-18180, 18194-18320,18335-18338, 18352-18395, 18409-18427, 18457-19016, 19030-19119,19133-19134, 19148-19217, 19231-19584, 19598-19748, 19762-19770,19784-19805, 19821-19881, 19896-19919, 19933-19944, 19958-19969,20004-20029, 20034-20289, 20303-20541, 20555-20559, 20573-20782,20796-20810, 20825-20860, 20874-21322, 21335-21377, 21390-21420,21431-21444, 21459-21626, 21629-21663, 21667-21841, 21856-21872,21886-21895, 21909-21937, 21957-22002, 22017-22209, 22223-22306,22320-22348, 22362-22637, 22651-22666, 22680-22747, 22761-22855,22869-23151, 23169-23203, 23218-23452, 23467-23735, 23749-23953,23967-23982, 23996-24014, 24028-24062, 24076-24121, 24136-24343,24358-24453, 24467-24475, 24489, 24503-24748, 24762-25030, 25044-25106,25120-25131, 25145-25227, 25241-25390, 25405-25407, 25436-25444,25458-25461, 25475-25578, 25653-25691, 25706-25720, 25748-25916,25944-25991, 26005-26093, 26113-26116, 26139-26178, 26194-26250,26308-26331, 26345-26362, 26376-26455, 26488-26514, 26528, 26560-26584,26598-26618, 26638-26642, 26656-26657, 26671-26703, 26717-26933,26954-26958, 26973-27216, 27238-27275, 27291, 27319-27372, 27391-27410,27425-27448, 27486-27600, 27615-27754, 27781-27943, 27957-27981,28011-28015, 28071-28098, 28114-28123, 28137-28235, 28257-28327,28349-28395, 28409-28505, 28520-28581, 28595-28614, 28628-28717,28731-28792, 28814-28843, 28857-28872, 28886-28900, 28915-28935,28955-28999, 29013-29229, 29256-29367, 29393-29438, 29452-29523,29537-29542, 29557-29656, 29671-29689, 29704-29761, 29784, 29808-29909,29925-30002, 30013-30020, 30025-30033, 30039-30067, 30074-30107,62210-62215, 62217-62225

SeqIDs w/o miR Seeds:

69, 73, 76, 79, 81-83, 86-88, 92, 94, 98, 101-102, 104-105, 107-113,117-120, 123, 132, 134-138, 141-142, 149-152, 156-159, 163-165, 167,174, 178, 183-185, 187-188, 190-191, 193-195, 200, 202-205, 208-211,213-217, 220, 225-226, 229, 231-234, 236, 238-239, 241-244, 248-251,253-261, 263-264, 266-280, 282-283, 285-286, 288-289, 293, 296-300,303-304, 306-308, 310-311, 313-318, 321, 323, 325-327, 329-336, 338,343, 346-347, 349, 352-354, 358, 360-368, 372-380, 382, 387-388, 391,393, 395-401, 404-409, 413, 415-417, 419-420, 422-425, 428, 431-432,434-438, 440-448, 450-457, 461, 464, 466, 468-471, 474, 476, 479-480,482-491, 493-494, 496-498, 501, 505-506, 508, 511-516, 518-519, 522,524-528, 530-531, 533, 537, 539-546, 548-553, 556-558, 562-567, 570-572,575, 577-582, 584-585, 587, 591-596, 600-605, 607, 609, 613-617,620-622, 626-627, 629, 631-633, 636, 638-644, 647-648, 651-654, 658-660,662-663, 665-675, 678-679, 681, 685-686, 688-693, 696, 698-699, 701,705-707, 711-717, 724-731, 738-742, 744-746, 748-750, 753-754, 756-757,759-765, 767, 770-772, 774, 777-781, 785, 787, 790-796, 800-814,818-819, 821-828, 830-832, 834-840, 847, 849-850, 852-860, 864, 868-871,873, 875, 877-879, 881-883, 885-888, 893-894, 897-898, 900, 903-904,906-911, 913-914, 919, 921-924, 927, 929-931, 934-939, 941, 943-944,946-949, 951-956, 958-959, 961-962, 964-965, 967-970, 974, 976-977,979-989, 991-992, 995-996, 998, 1001-1013, 1015-1017, 1019-1021,1026-1034, 1036, 1038, 1040-1049, 1051-1053, 1055, 1057, 1059-1061,1063-1066, 1069-1074, 1077, 1081-1082, 1084-1085, 1087, 1089, 1094-1095,1097-1104, 1106, 1108, 1110-1116, 1120-1122, 1124-1127, 1130, 1134,1136-1138, 1140-1143, 1145, 1149, 1151, 1154-1163, 1165-1169, 1171,1174-1175, 1177, 1179-1181, 1183-1187, 1189, 1191-1194, 1196-1204, 1206,1209-1210, 1212-1214, 1216-1217, 1221-1222, 1224-1236, 1238, 1240-1241,1243-1252, 1255-1256, 1258, 1260, 1262-1265, 1267, 1270, 1272-1275,1277-1283, 1285-1288, 1291-1300, 1302, 1304-1305, 1307, 1309-1314,1316-1322, 1324-1326, 1330-1332, 1334-1335, 1341-1342, 1344-1351,1353-1365, 1367, 1369-1370, 1373-1375, 1378-1388, 1390-1392, 1395-1398,1401-1402, 1406-1420, 1422, 1425, 1428-1429, 1431, 1434-1435, 1439,1442, 1446, 1448, 1453-1455, 1457, 1459-1469, 1471, 1474-1475, 1477,1480-1482, 1484-1488, 1490-1493, 1498, 1500-1503, 1505-1511, 1513, 1515,1520, 1522, 1524-1530, 1533, 1537, 1540-1547, 1550-1551, 1553-1554,1557-1562, 1564, 1567, 1569, 1571-1572, 1574-1578, 1580-1582, 1584-1585,1587-1589, 1591-1593, 1596-1598, 1601-1604, 1607-1608, 1610-1617,1620-1622, 1625-1631, 1633, 1635-1637, 1640-1641, 1643, 1645-1646, 1648,1650-1651, 1655, 1657, 1659-1662, 1664-1667, 1669, 1671-1675, 1680,1682-1683, 1688-1692, 1694, 1696-1698, 1700-1703, 1705, 1707-1713,1715-1716, 1718-1720, 1723, 1727, 1730-1731, 1734, 1738-1741, 1743,1745-1746, 1748, 1750-1751, 1753, 1755-1760, 1762-1766, 1768-1772, 1774,1776, 1780, 1784, 1786-1788, 1791, 1793, 1796, 1799-1802, 1805,1807-1811, 1813, 1815, 1817, 1819-1822, 1824-1826, 1832-1838, 1840,1842, 1844-1849, 1851, 1853-1854, 1858, 1862, 1864, 1867, 1870,1872-1873, 1875, 1878, 1880-1884, 1891, 1893-1899, 1901-1905, 1907,1911-1912, 1916-1920, 1924-1925, 1929, 1931-1932, 1934-1937, 1939-1940,1943-1947, 1949-1954, 1958-1959, 1962, 1964-1965, 1967-1970, 1973-1974,1976-1977, 1979-1980, 1982, 1984-1985, 1987, 1989, 1992, 1995-1996,1998, 2003, 2005, 2007, 2010-2012, 2014-2016, 2018-2022, 2024-2025,2030, 2032-2033, 2037-2039, 2043-2047, 2051-2056, 2062, 2065-2066,2074-2075, 2077-2083, 2085-2086, 2089-2090, 2094-2099, 2104-2111, 2113,2116-2119, 2121-2123, 2125-2127, 2130-2135, 2137-2140, 2142, 2145-2146,2149-2154, 2156-2167, 2169-2170, 2172-2178, 2180-2181, 2183-2184,2186-2188, 2190, 2192, 2195-2196, 2198-2199, 2202-2203, 2207-2211,2213-2222, 2225-2228, 2231-2236, 2238-2244, 2246, 2248, 2250-2251,2256-2258, 2262-2264, 2266, 2268, 2272-2276, 2279, 2281-2283, 2285-2288,2292-2297, 2299, 2302-2310, 2312-2313, 2315-2318, 2320-2331, 2335, 2337,2340, 2345-2347, 2350-2352, 2354-2355, 2360, 2362-2363, 2366-2376, 2378,2381-2387, 2389, 2391, 2393-2398, 2400-2408, 2410-2411, 2413-2417, 2419,2425-2432, 2434-2439, 2445, 2447, 2449-2451, 2453-2459, 2463-2467,2470-2473, 2475-2476, 2478-2481, 2483-2501, 2503-2505, 2509, 2511-2514,2517-2518, 2522, 2524-2525, 2527, 2530, 2533-2535, 2537-2539, 2542-2552,2554-2555, 2557, 2560, 2562-2568, 2572-2573, 2576-2577, 2579, 2581-2582,2584, 2586, 2588-2589, 2591-2592, 2594-2600, 2603-2605, 2611-2613,2615-2618, 2620-2624, 2626, 2628-2629, 2631, 2634-2639, 2644-2647,2649-2656, 2659-2660, 2662-2667, 2669-2676, 2679-2682, 2684-2685,2688-2691, 2693-2694, 2696-2699, 2703, 2705-2711, 2713-2731, 2734-2735,2737-2740, 2742, 2744-2746, 2749-2755, 2759, 2761-2763, 2766-2769, 2771,2773-2775, 2777, 2779-2781, 2783-2784, 2786-2787, 2791, 2795-2796, 2804,2806-2808, 2810, 2812-2813, 2815, 2817-2818, 2826-2827, 2830-2840,2843-2844, 2850-2857, 2859-2861, 2867, 2869-2872, 2876-2877, 2879-2880,2885-2887, 2889, 2895-2897, 2899-2903, 2911, 2913-2917, 2919-2920, 2923,2925-2930, 2933-2938, 2943, 2950-2954, 2956-2958, 2960-2961, 2963-2965,2970-2971, 2973, 2975-2976, 2978-2980, 2983-2988, 2990-2993, 2995-3010,3012-3016, 3019, 3021-3023, 3025, 3028-3029, 3032, 3034-3038, 3040,3043-3049, 3051-3053, 3058-3063, 3066-3069, 3071, 3076-3077, 3079-3082,3085-3093, 3095-3097, 3099-3101, 3104-3106, 3108-3109, 3111-3112,3114-3115, 3118-3119, 3121-3123, 3128-3129, 3131, 3133-3140, 3144, 3146,3148-3156, 3162, 3164, 3166, 3168, 3171-3173, 3175-3176, 3178,3180-3181, 3183-3186, 3188-3189, 3193-3194, 3197, 3199-3204, 3207-3208,3210-3212, 3214, 3216, 3219, 3221-3223, 3225-3229, 3232-3234, 3236,3239, 3241-3242, 3245-3251, 3253-3257, 3259-3265, 3268-3269, 3272-3279,3283-3285, 3287-3288, 3290, 3293-3295, 3299-3301, 3303, 3306-3308, 3312,3314-3318, 3324, 3326-3333, 3336, 3338-3340, 3342-3348, 3350, 3352-3355,3357-3377, 3379-3381, 3383-3385, 3387, 3392-3394, 3397-3398, 3401, 3407,3409, 3411-3412, 3414, 3418-3424, 3428, 3432, 3436-3437, 3439-3440,3442, 3445, 3455-3457, 3459, 3461-3465, 3468, 3474, 3478-3479, 3481,3483, 3485-3487, 3491-3492, 3494-3500, 3502-3508, 3512, 3515, 3521-3530,3532-3535, 3537-3538, 3540-3541, 3543, 3546, 3548-3551, 3553, 3555-3557,3559, 3561-3562, 3565-3567, 3572, 3574, 3576, 3579, 3581, 3584,3589-3590, 3594-3595, 3597-3599, 3602-3606, 3609-3614, 3616, 3618,3624-3631, 3633-3635, 3637-3639, 3643, 3646, 3649, 3651-3652, 3655-3665,3669, 3672-3673, 3675, 3677-3678, 3680-3681, 3683-3687, 3689, 3692,3697-3699, 3701, 3703, 3707-3712, 3716, 3718, 3723-3724, 3726-3731,3734-3735, 3739-3740, 3743-3748, 3751, 3753-3754, 3756, 3758, 3763-3768,3772, 3774-3777, 3779-3780, 3782-3783, 3786-3790, 3792, 3794-3799,3801-3802, 3804, 3806, 3808-3813, 3815, 3817-3821, 3824, 3827-3830,3832-3833, 3836-3837, 3840-3843, 3846, 3849-3850, 3852-3858, 3860, 3862,3864-3873, 3875-3885, 3887, 3890-3891, 3893, 3896-3899, 3901, 3904,3908-3912, 3914-3916, 3920, 3923-3929, 3931-3932, 3934, 3937-3945,3947-3948, 3950, 3953, 3956-3958, 3961, 3964, 3969, 3971-3975, 3977,3979-3981, 3983, 3985-3990, 3995, 3997-3999, 4001-4002, 4004, 4006-4010,4014, 4016, 4020-4023, 4027, 4030-4039, 4041, 4043, 4045-4047, 4049,4051, 4053-4055, 4058, 4060-4064, 4066, 4068-4077, 4079-4081, 4083,4085-4086, 4088-4090, 4092-4093, 4095-4096, 4099-4100, 4103-4105,4107-4108, 4110-4111, 4116-4117, 4119-4120, 4124-4125, 4131-4134, 4136,4138, 4142-4149, 4156-4169, 4173, 4176-4177, 4179-4182, 4184-4189,4191-4193, 4195-4199, 4202, 4204-4213, 4215, 4217, 4221, 4223-4226,4229, 4231-4232, 4234-4235, 4237, 4239, 4241, 4244-4247, 4249-4252,4255-4256, 4259, 4265, 4268-4271, 4273-4278, 4280-4281, 4283-4285,4287-4297, 4299, 4301-4302, 4304-4310, 4312-4317, 4319-4325, 4327-4328,4330, 4332-4338, 4340-4341, 4344-4349, 4353, 4355-4364, 4366-4368,4370-4374, 4377-4379, 4383, 4387-4390, 4393-4398, 4401-4402, 4404-4411,4413, 4417-4424, 4427-4428, 4430-4431, 4433-4434, 4437-4439, 4441-4446,4448, 4454-4455, 4457, 4459, 4461-4462, 4464-4465, 4468-4475, 4477,4479-4481, 4483-4484, 4486, 4488-4489, 4493-4495, 4500, 4502-4510,4513-4522, 4524-4530, 4532-4535, 4537, 4539-4544, 4546-4550, 4552,4557-4558, 4561-4565, 4569-4570, 4573, 4576-4580, 4582-4591, 4593-4600,4602-4604, 4606-4607, 4611, 4615-4616, 4618-4620, 4622, 4627, 4629-4631,4633-4635, 4637-4638, 4640, 4643-4644, 4646-4650, 4652, 4654-4655,4658-4660, 4662-4663, 4666-4668, 4670-4672, 4680-4682, 4684, 4686-4690,4692-4697, 4699-4700, 4702-4706, 4708-4710, 4712-4713, 4715-4718,4720-4721, 4723-4727, 4729, 4739-4745, 4747-4750, 4753, 4755-4760,4762-4764, 4766-4767, 4769-4770, 4772-4775, 4777-4778, 4782, 4785-4792,4795-4797, 4799, 4801-4802, 4804-4806, 4809-4817, 4819-4824, 4826-4829,4832-4837, 4839-4847, 4849-4853, 4855-4861, 4863, 4865-4882, 4885-4886,4888, 4892-4901, 4903, 4905, 4909-4922, 4925-4927, 4931, 4933-4934,4936-4938, 4941, 4943-4944, 4946-4947, 4955, 4957-4961, 4964, 4966,4968-4976, 4978, 4981, 4986-4991, 4993, 5000-5003, 5006, 5008-5009,5012-5013, 5015-5020, 5022-5024, 5026-5031, 5033, 5035, 5037, 5039-5041,5047-5050, 5054-5055, 5057-5058, 5060, 5062-5064, 5067-5073, 5075,5077-5081, 5083, 5085-5086, 5090-5095, 5097-5098, 5100-5101, 5103,5105-5107, 5109-5110, 5112-5113, 5116, 5120-5122, 5124, 5126, 5129,5131, 5134-5138, 5140-5143, 5146-5159, 5161-5163, 5165-5166, 5168-5171,5174, 5176, 5178-5182, 5185-5188, 5190-5191, 5193-5195, 5197, 5199,5201-5203, 5207, 5212, 5214-5215, 5218-5219, 5221, 5223-5224, 5237-5238,5242-5244, 5246-5248, 5251-5258, 5261, 5263-5265, 5268-5274, 5276-5279,5281-5282, 5284-5286, 5288, 5291, 5293, 5295-5304, 5306, 5308,5310-5314, 5320-5323, 5325-5326, 5328, 5330-5337, 5341-5346, 5351-5369,5372, 5375-5377, 5383-5386, 5389-5391, 5393-5397, 5399, 5402-5407,5411-5413, 5419-5427, 5429, 5431, 5433-5434, 5436-5437, 5441-5443,5445-5451, 5453-5454, 5456, 5459, 5461-5465, 5467-5473, 5479-5481,5483-5485, 5488, 5490, 5492-5493, 5496-5497, 5499-5503, 5507, 5510-5512,5515, 5518-5521, 5523-5524, 5526-5527, 5529, 5531, 5534-5535, 5537,5540-5542, 5544, 5546, 5548, 5550, 5552, 5554-5558, 5560-5561,5563-5566, 5570, 5572-5573, 5577-5579, 5581, 5583-5587, 5590-5592, 5595,5599-5617, 5627, 5630, 5632-5633, 5635, 5637-5638, 5641-5643, 5645,5647-5651, 5653-5655, 5657-5662, 5664-5665, 5669, 5671, 5673, 5675,5677, 5680, 5682-5683, 5685, 5687-5688, 5690, 5692, 5695-5699, 5702,5707, 5709-5712, 5714, 5717-5718, 5720-5722, 5725, 5727-5729, 5732-5741,5743-5749, 5752-5755, 5757-5765, 5769-5771, 5773, 5775-5776, 5778-5779,5782, 5785-5792, 5794, 5797-5799, 5801-5802, 5804-5808, 5811-5812, 5814,5819, 5821-5830, 5832-5839, 5844-5845, 5848, 5850-5851, 5853-5856, 5858,5860-5870, 5872, 5874-5875, 5879-5883, 5888-5891, 5893, 5896-5898,5900-5901, 5903-5905, 5910-5913, 5916-5926, 5929, 5932-5939, 5941, 5944,5947, 5950-5954, 5956-5957, 5959-5960, 5963-5965, 5969-5973, 5976,5978-5980, 5982, 5984-5986, 5988-5992, 5997-6002, 6004-6006, 6008-6010,6012-6017, 6019-6027, 6030, 6034-6036, 6040, 6048-6049, 6052-6059,6061-6062, 6064, 6066-6069, 6071-6075, 6077, 6079, 6082-6084, 6086-6091,6093-6094, 6096, 6098-6100, 6106, 6111-6114, 6117, 6125, 6127, 6129,6131, 6133-6135, 6137-6140, 6144, 6151, 6153, 6157, 6159, 6163, 6166,6169, 6173-6174, 6176-6181, 6187, 6189, 6191-6194, 6197, 6200-6204,6206, 6208-6210, 6213, 6215-6220, 6225-6226, 6232, 6235, 6237-6238,6240-6241, 6245, 6248-6249, 6251, 6253, 6255-6257, 6260-6265, 6269,6274-6276, 6278, 6282-6283, 6285-6286, 6288-6290, 6294-6297, 6299,6301-6304, 6306-6308, 6310-6311, 6314-6322, 6324-6333, 6335-6337, 6340,6342-6347, 6350-6353, 6356, 6361-6362, 6364-6368, 6370, 6373-6385,6387-6400, 6402-6408, 6413, 6415, 6417-6419, 6425-6437, 6443-6444, 6446,6451-6454, 6456-6462, 6464-6466, 6469, 6471-6472, 6475-6477, 6481-6485,6493, 6496, 6498, 6504-6508, 6511, 6513-6522, 6525-6527, 6529-6532,6534-6550, 6553, 6555-6558, 6561, 6567, 6572-6574, 6576-6578, 6580-6581,6584, 6587, 6589-6593, 6595, 6599, 6601-6608, 6610-6616, 6619, 6622,6624-6625, 6629-6630, 6632-6634, 6637-6638, 6640, 6642-6645, 6648-6649,6651, 6653-6663, 6665-6673, 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28734,28737-28740, 28743-28747, 28750, 28752, 28755-28760, 28763-28769, 28771,28773-28779, 28781-28788, 28790, 28792-28796, 28799-28800, 28808,28811-28812, 28815, 28817, 28819, 28821, 28825-28828, 28832,28838-28845, 28847-28850, 28854, 28856, 28858, 28867, 28869-28873,28875, 28881-28885, 28887-28892, 28894, 28899-28906, 28908-28909,28911-28920, 28923, 28926, 28929-28930, 28932, 28934-28936, 28940,28943-28944, 28946, 28949-28951, 28954, 28957-28959, 28961, 28963,28965, 28970, 28972-28981, 28984-28988, 28990, 28993-28994, 28996,28998-28999, 29004-29005, 29009-29010, 29013-29021, 29023, 29025-29028,29032-29033, 29035-29036, 29038-29041, 29044, 29046-29055, 29057-29058,29062, 29064-29066, 29068-29073, 29075-29084, 29087, 29089-29094,29097-29098, 29100, 29103, 29106, 29108, 29110-29114, 29117-29118,29120-29121, 29124, 29127, 29129-29130, 29132-29133, 29137, 29139-29140,29142-29143, 29145-29150, 29152-29158, 29161, 29163-29168, 29170-29179,29182, 29185, 29188-29189, 29193, 29195-29196, 29199-29200, 29202-29212,29214-29215, 29217-29218, 29220-29222, 29224-29229, 29231, 29235, 29241,29247-29248, 29250, 29252-29259, 29261, 29265, 29268-29273, 29275-29281,29283-29284, 29286-29287, 29289-29297, 29299, 29302-29304, 29306-29309,29311-29313, 29315-29320, 29322-29324, 29326, 29328-29331, 29333-29338,29340-29342, 29345, 29347-29364, 29371, 29373-29376, 29379-29380,29384-29386, 29391-29397, 29399, 29402-29406, 29408, 29410-29413, 29417,29419-29422, 29425-29431, 29437, 29440-29441, 29444, 29446, 29449-29451,29453-29454, 29456, 29458-29461, 29464, 29466-29476, 29478, 29480-29483,29485, 29487-29496, 29498-29500, 29502-29504, 29506, 29508, 29510-29511,29513, 29516-29522, 29526, 29528, 29530-29532, 29536, 29538-29539,29541-29542, 29547, 29549-29552, 29555, 29557-29566, 29569-29577,29579-29584, 29586-29592, 29597-29598, 29601-29602, 29604, 29606-29608,29610-29612, 29614-29618, 29620, 29622-29623, 29625, 29628-29633,29635-29636, 29638-29640, 29646, 29649-29650, 29655-29656, 29661,29663-29670, 29675-29676, 29679, 29681-29685, 29689, 29696, 29699-29701,29703-29705, 29707-29709, 29711-29713, 29715-29719, 29721-29722,29724-29728, 29735-29747, 29749-29753, 29755-29756, 29760-29761, 29763,29769, 29771, 29776-29785, 29789, 29791, 29793, 29800, 29802-29803,29808-29810, 29812-29813, 29816, 29818-29821, 29823, 29825-29827,29830-29831, 29833-29843, 29846, 29848-29854, 29856, 29858-29859,29861-29866, 29869-29870, 29876, 29879, 29881, 29884, 29886-29891,29893, 29896-29897, 29899-29908, 29910, 29912-29913, 29916-29918, 29922,29924-29939, 29941, 29943-29945, 29947-29950, 29952-29953, 29955-29956,29958, 29960-29961, 29963, 29965-29968, 29972-29973, 29975-29976,29978-29983, 29986-29990, 29993, 29995, 29997-30000, 30002, 30005-30006,30011, 30013-30016, 30020, 30022-30024, 30028, 30037, 30039-30041,30046-30055, 30057, 30059-30062, 30071, 30073, 30078, 30080,30083-30084, 30087, 30089, 30093-30094, 30096, 30098, 30101-30103,30105, 62210, 62213, 62216, 62218-62219, 62221-62223

Single Strand Oligonucleotides (Sense Strand of Target Gene BDNF)

SegID range: 30003-62209

SegIDs w/o G Runs:

30013-30020, 30025-30033, 30039-30067, 30074-30433, 30447-30469,30491-30555, 30569-30591, 30605-30678, 30688-30906, 30922-31097,31111-31168, 31182-31331, 31345-31392, 31406-31527, 31543-31718,31732-31879, 31893-32056, 32070-32497, 32511-33009, 33024-33121,33135-33939, 33953-34029, 34044-34326, 34340-34657, 34671-35030,35045-35107, 35111-35348, 35362-35370, 35384-35432, 35446-35767,35781-35811, 35826-36244, 36259-36875, 36889-37145, 37159-37172,37187-37197, 37211-37222, 37237-37255, 37269-37288, 37302-37644,37658-37827, 37841-37901, 37915-37960, 37974-38022, 38036-38058,38073-38145, 38159-38163, 38175-38240, 38253-38353, 38367-38763,38773-38811, 38825-38887, 38898, 38912-38954, 38969-38986, 39000-39084,39098-39177, 39193-39425, 39439-39486, 39501-39551, 39574-39584,39599-39663, 39677-39690, 39704-39717, 39732-39839, 39853-39868,39883-40073, 40095-40205, 40219-40270, 40284-40397, 40422-40519,40534-40687, 40711-40775, 40796, 40810-40956, 40970-41642, 41656-41660,41674-41675, 41689-41730, 41744-41782, 41796-41894, 41913-42057,42071-42147, 42161-42176, 42190, 42204-42234, 42250-42320, 42334-42354,42368-42369, 42383-42415, 42429-42784, 42808-42849, 42864-43023,43037-43058, 43072-43143, 43158-43479, 43493-43531, 43545-44409,44423-44817, 44831-44892, 44906-45185, 45199-45281, 45295-45317,45331-45352, 45371-45395, 45409-45480, 45489-45544, 45551-45895,45909-46075, 46089-46145, 46159-46176, 46190, 46205-46207, 46221-46238,46257-46270, 46291-46299, 46313-46417, 46431-46493, 46514-46559,46573-46616, 46636-46697, 46712-46754, 46768-46782, 46796-46800,46815-46865, 46879-46934, 46948-46980, 46994-47090, 47105-47447,47461-47538, 47552-47591, 47606-47801, 47815-47819, 47833-47878,47894-48512, 48526-48582, 48596-48649, 48664-48836, 48850-49135,49149-49762, 49776-49824, 49838-49879, 49906-49995, 50009-50260,50274-50356, 50370-50382, 50396-50801, 50815-51308, 51322-51577,51592-51613, 51632-52026, 52040-52111, 52125-52144, 52158-52261,52275-52462, 52476-52477, 52515-52623, 52638-52650, 52677-52710,52725-52816, 52859-52878, 52892-52921, 52935-52958, 52986-53019,53034-53058, 53079-53096, 53110-53141, 53156-53161, 53175-53264,53278-53300, 53324-53333, 53347-53351, 53400-53407, 53422-53441,53455-53571, 53585-53597, 53622-53634, 53649-53660, 53674-53699,53714-53732, 53747-53760, 53775-53803, 53833-53908, 53928-53929,53943-54010, 54026-54316, 54330-54383, 54406-54532, 54546-54555,54572-54595, 54621, 54635-54760, 54774-54842, 54856-55244, 55258-55289,55303-55417, 55432-56304, 56318-56327, 56342-56714, 56730-56771,56786-57271, 57302-57523, 57537-57592, 57615-57619, 57633-57950,57963-58064, 58080-58093, 58108-58111, 58125-58148, 58176-58201,58216-58281, 58313-58321, 58335-58395, 58409-58468, 58482-58662,58676-58689, 58704-58735, 58749-58767, 58798-58830, 58844-58858,58876-58880, 58955-59196, 59210-59326, 59353, 59367-59455, 59480-59637,59664-59743, 59767-59769, 59784-59788, 59804-60924, 60938-60950,60964-60993, 61007-61060, 61075-61088, 61102-61137, 61168-61220,61247-61292, 61306-61354, 61368-61392, 61423-61432, 61446-61609,61623-61689, 61724-61725, 61739-61779, 61795-61797, 61847-61850,61866-61914, 61928-62029, 62068-62070, 62094-62100, 62114-62145,62159-62209

SeqIDs w/o miR Seeds:

30005-30006, 30011, 30013-30016, 30020, 30022-30024, 30028, 30037,30039-30041, 30046-30055, 30057, 30059-30062, 30071, 30073, 30078,30080, 30083-30084, 30087, 30089, 30093-30094, 30096, 30098,30101-30103, 30105, 30108-30109, 30112, 30114-30117, 30119-30120, 30126,30133, 30136-30137, 30139-30148, 30153-30158, 30160-30164, 30166-30169,30171-30172, 30176-30178, 30180-30181, 30183-30186, 30189-30191,30195-30198, 30200-30201, 30204-30210, 30213-30214, 30216-30218,30221-30222, 30225-30228, 30230-30232, 30235-30240, 30242, 30244-30252,30254, 30256-30259, 30262-30268, 30271, 30274, 30276-30280, 30282-30283,30286-30297, 30299-30302, 30304-30305, 30307-30308, 30310-30314,30316-30318, 30322-30337, 30339-30340, 30342, 30345, 30347-30353,30355-30356, 30358-30360, 30362-30367, 30369-30375, 30379, 30381-30385,30387-30390, 30393-30394, 30396-30397, 30399-30400, 30410-30416,30418-30426, 30428-30429, 30433-30436, 30438, 30444, 30447-30453,30455-30458, 30460-30461, 30464-30468, 30473-30475, 30479-30483, 30485,30490-30492, 30495-30504, 30506, 30508-30509, 30511-30514, 30516, 30518,30520, 30524-30529, 30531-30541, 30544, 30547-30549, 30552, 30554-30555,30558-30567, 30569-30570, 30572-30573, 30575-30581, 30586-30587,30589-30590, 30592-30600, 30602, 30607, 30610, 30616, 30618-30619,30622-30623, 30625, 30627, 30629-30633, 30635-30636, 30638, 30640-30642,30646, 30650-30652, 30655, 30657-30658, 30660-30664, 30666-30667,30670-30671, 30674, 30677-30679, 30681, 30683, 30685-30686, 30688,30690, 30694, 30697, 30699, 30702-30703, 30705-30706, 30709,30711-30714, 30716-30717, 30719, 30722-30723, 30725, 30727, 30729-30730,30732, 30734-30738, 30742-30752, 30757-30758, 30761, 30764-30765, 30767,30769, 30772-30776, 30778-30782, 30784-30786, 30788-30792, 30794-30795,30797, 30800-30801, 30806-30811, 30813-30814, 30821-30828, 30831,30833-30839, 30842-30849, 30851, 30853, 30856-30857, 30859-30870,30872-30874, 30878, 30880-30882, 30884-30894, 30896-30908, 30911,30913-30915, 30921, 30923-30926, 30928-30929, 30931-30933, 30935-30938,30940-30942, 30945-30946, 30949-30950, 30952-30961, 30963, 30967-30972,30974-30981, 30983-30988, 30990-30999, 31002-31003, 31007, 31011-31015,31017-31020, 31022-31023, 31025-31029, 31031, 31033, 31038, 31040,31042-31049, 31051, 31053-31055, 31057, 31059-31064, 31066-31067, 31069,31071, 31073-31074, 31076, 31080-31081, 31083-31091, 31093-31094,31096-31099, 31101-31104, 31106, 31113, 31116, 31118, 31124-31125,31127-31128, 31130, 31132-31134, 31136-31137, 31141, 31143, 31147-31148,31150, 31152-31157, 31160-31161, 31166-31167, 31169-31170, 31173-31174,31180, 31183-31185, 31188, 31191, 31193-31196, 31199, 31203-31209,31211, 31213, 31215-31216, 31218-31220, 31222, 31225-31227, 31229-31232,31234, 31236-31240, 31243-31247, 31249, 31251-31253, 31256-31262,31264-31268, 31270-31272, 31274, 31277-31282, 31284-31285, 31289, 31291,31293-31300, 31302-31303, 31305-31309, 31312, 31314, 31320, 31322-31323,31327, 31329, 31332-31336, 31338-31339, 31341, 31343-31350, 31352-31358,31361-31366, 31368-31372, 31378-31379, 31382-31386, 31388, 31390-31425,31428-31431, 31433, 31435, 31438-31439, 31441-31446, 31448-31453, 31457,31459, 31466-31469, 31472, 31475-31484, 31486, 31488-31489, 31493-31497,31499, 31502-31503, 31505-31513, 31515, 31517-31524, 31526-31528, 31530,31534, 31539, 31541-31542, 31545-31546, 31549-31550, 31552-31557, 31559,31563-31564, 31566, 31569-31570, 31572-31580, 31583-31588, 31590-31593,31595-31614, 31616, 31618-31625, 31627-31629, 31632, 31634, 31638,31640-31644, 31647-31649, 31651-31652, 31655, 31659-31663, 31665-31668,31673, 31675-31682, 31685-31693, 31695, 31698-31704, 31706-31707, 31709,31711-31721, 31726, 31730-31734, 31737-31741, 31745, 31749-31756,31758-31761, 31763-31771, 31773, 31775-31778, 31780-31781, 31787-31788,31791-31798, 31800-31801, 31804-31807, 31809-31812, 31814-31815,31817-31820, 31822-31834, 31838-31839, 31841-31842, 31844-31845,31847-31851, 31853, 31855-31856, 31858-31861, 31864-31865, 31869-31870,31873, 31875-31876, 31879-31888, 31893-31895, 31897, 31899-31900, 31903,31905-31906, 31908-31912, 31914-31918, 31921-31923, 31925, 31927,31929-31932, 31934-31945, 31947-31948, 31950-31951, 31954, 31958, 31960,31962, 31964-31966, 31968, 31973, 31975-31977, 31979-31981, 31987-31991,31993, 31996-31998, 32000-32003, 32005-32006, 32010-32020, 32022-32024,32026-32030, 32036, 32039-32043, 32045-32051, 32053-32060, 32062-32064,32066, 32070-32077, 32083-32084, 32086-32087, 32089, 32091-32093,32095-32104, 32106-32107, 32110, 32113-32114, 32117-32122, 32124-32126,32130-32131, 32133-32134, 32137, 32139, 32142, 32144-32145, 32149,32156, 32158-32159, 32161, 32165-32168, 32171-32178, 32181, 32183-32186,32188-32203, 32205-32206, 32208, 32210-32213, 32215-32216, 32218,32220-32222, 32224, 32226, 32228-32238, 32240-32242, 32244-32246,32249-32254, 32259-32263, 32265-32266, 32268-32269, 32271-32275,32277-32278, 32281-32293, 32295-32300, 32302, 32307-32312, 32315, 32318,32320, 32322-32329, 32331-32333, 32340-32345, 32348-32354, 32356-32358,32360-32366, 32369, 32371-32376, 32378-32388, 32392, 32394-32398,32406-32411, 32414, 32420-32421, 32426, 32429-32432, 32435-32441,32444-32449, 32451-32452, 32454, 32456-32458, 32460, 32462, 32464-32465,32467-32468, 32472, 32475-32476, 32478, 32480-32483, 32485, 32488,32490-32492, 32494-32495, 32497-32501, 32503-32505, 32507, 32509-32512,32515, 32517, 32519-32528, 32530-32534, 32537-32544, 32547, 32549-32550,32552-32553, 32556-32561, 32563-32565, 32567-32568, 32571-32576,32578-32579, 32581-32583, 32585, 32587, 32589-32590, 32593, 32596-32598,32600, 32602-32603, 32605-32610, 32613-32622, 32624, 32626-32627,32629-32630, 32632-32634, 32636, 32640-32643, 32645, 32647-32648, 32651,32656-32658, 32660-32662, 32664-32666, 32668-32681, 32683-32684, 32686,32690-32691, 32699-32701, 32704-32705, 32707-32714, 32718, 32720,32722-32725, 32727-32732, 32734, 32736-32740, 32742-32743, 32747, 32750,32754, 32756-32758, 32760-32763, 32765-32767, 32769-32780, 32784,32786-32795, 32799-32803, 32805, 32808-32813, 32815-32816, 32818, 32820,32822, 32824, 32826, 32828, 32830-32832, 32834-32836, 32838-32839,32841-32844, 32846, 32848-32850, 32853-32857, 32860-32863, 32865-32866,32869, 32871-32875, 32878-32880, 32884-32891, 32893-32894, 32896-32897,32899-32901, 32904-32906, 32908, 32910-32914, 32917, 32919-32922, 32927,32930-32933, 32938, 32940-32942, 32944-32948, 32951-32955, 32957-32960,32962-32963, 32966-32968, 32971-32978, 32980-32982, 32984, 32988-32991,32995-32998, 33001, 33006, 33009-33014, 33016-33017, 33020, 33024-33025,33027, 33031-33032, 33034, 33036-33039, 33041, 33043, 33047-33048,33050-33061, 33063, 33065, 33068, 33071-33074, 33076-33078, 33080-33082,33084, 33086-33087, 33089-33092, 33094, 33096, 33098, 33100-33102,33104, 33106-33110, 33112-33117, 33119-33122, 33124-33127, 33129, 33132,33138-33145, 33147, 33149-33154, 33156, 33158-33160, 33162-33163,33165-33167, 33169-33176, 33178, 33181-33183, 33186, 33190-33191, 33193,33195-33201, 33205-33209, 33211-33214, 33216, 33220-33225, 33228-33230,33232-33241, 33247-33248, 33250-33251, 33253-33254, 33256-33261, 33264,33266, 33268, 33271-33273, 33275-33283, 33286-33291, 33294-33298, 33300,33303-33307, 33311-33312, 33314-33317, 33319-33321, 33324-33325,33327-33329, 33331, 33333, 33335, 33338-33341, 33343, 33345-33346,33348-33349, 33351, 33354-33356, 33358, 33361, 33363, 33368-33370,33372-33374, 33376, 33380-33382, 33384, 33386, 33389, 33391-33394,33396-33403, 33405-33407, 33412, 33414-33416, 33418-33420, 33422-33423,33425-33427, 33429, 33431, 33437, 33440-33442, 33444, 33446, 33448,33451, 33453, 33455, 33457-33463, 33465, 33467, 33469-33475,33479-33484, 33487, 33489-33491, 33495-33502, 33504-33508, 33517-33520,33522-33523, 33526-33527, 33530-33531, 33533-33540, 33543-33553,33557-33559, 33561-33563, 33566-33575, 33577, 33580-33585, 33587,33589-33596, 33599-33615, 33617-33619, 33622-33623, 33625, 33628-33636,33640, 33644-33646, 33648-33649, 33653-33654, 33657-33661, 33664-33665,33668, 33670-33678, 33680-33682, 33684-33687, 33689, 33691-33699,33706-33707, 33709-33711, 33717-33725, 33727, 33729-33731, 33733-33736,33738, 33741, 33743, 33745-33747, 33750, 33753, 33755-33756, 33758,33760, 33762-33765, 33767-33768, 33770-33771, 33773-33775, 33777-33790,33792-33794, 33796, 33798-33800, 33804, 33807, 33809, 33811-33812,33814-33820, 33822-33823, 33827-33828, 33833-33835, 33840-33846, 33848,33850-33851, 33856-33857, 33859-33861, 33863-33864, 33866-33868,33870-33872, 33874-33881, 33883-33884, 33886-33887, 33896-33898,33900-33902, 33904-33906, 33909-33910, 33912-33915, 33919, 33923-33926,33928, 33931-33935, 33937-33938, 33940-33941, 33944-33946, 33948-33949,33953-33954, 33956-33958, 33960, 33962-33963, 33965-33967, 33971-33977,33979-33980, 33982, 33984, 33986-33989, 33992, 33994, 33998,34001-34003, 34005-34008, 34010-34011, 34013-34014, 34016, 34018-34020,34025-34026, 34029, 34031, 34033, 34037-34038, 34040, 34044-34048,34050-34064, 34066-34068, 34070-34078, 34080-34082, 34084, 34086, 34088,34090, 34093, 34096, 34098, 34104, 34107-34111, 34116, 34118,34121-34124, 34127-34129, 34132-34138, 34140-34142, 34144, 34148-34154,34157, 34159, 34161, 34164, 34166-34167, 34169, 34171, 34173-34175,34177-34178, 34180-34186, 34188-34202, 34204, 34207-34208, 34210,34212-34213, 34215-34216, 34218, 34223, 34226-34233, 34235-34236,34238-34240, 34243, 34246, 34249-34253, 34255-34260, 34262-34266,34268-34270, 34274-34282, 34285-34289, 34292-34293, 34296, 34301,34303-34307, 34309-34312, 34316-34330, 34333-34334, 34336-34337, 34340,34342, 34344-34345, 34347, 34351-34358, 34360, 34362-34364, 34366-34369,34371, 34373-34374, 34377-34385, 34387, 34391, 34394, 34396-34397,34399-34405, 34407, 34412-34416, 34422-34433, 34437, 34440-34447,34450-34451, 34457-34458, 34460-34461, 34463-34465, 34467-34471,34473-34475, 34481-34484, 34488, 34490-34493, 34496-34497, 34499, 34501,34503-34511, 34513, 34515-34517, 34520, 34522-34523, 34525-34527,34531-34532, 34534-34537, 34540, 34542, 34544, 34546, 34548-34549,34554, 34556, 34558, 34560, 34562, 34565-34566, 34568-34570, 34572,34574, 34576, 34581-34582, 34585-34589, 34591, 34595, 34597-34598,34600-34601, 34604-34605, 34608, 34611-34614, 34616, 34618-34622,34624-34625, 34627, 34630, 34633-34634, 34636, 34638, 34640-34641,34647-34650, 34652-34653, 34659-34660, 34663-34666, 34668-34669, 34671,34673-34676, 34678, 34680-34681, 34685-34687, 34689-34690, 34694-34697,34699-34701, 34703-34706, 34709-34719, 34725, 34728, 34731-34734,34736-34745, 34748-34754, 34759, 34762-34766, 34768-34769, 34772, 34774,34777, 34782, 34784, 34786, 34790-34791, 34796-34798, 34800, 34804,34806-34808, 34810-34812, 34814-34816, 34821-34823, 34825-34826,34828-34832, 34834-34838, 34840-34841, 34843-34846, 34850-34854,34856-34864, 34867-34870, 34872-34873, 34875, 34881-34886, 34888-34896,34902, 34906-34918, 34920, 34923, 34926-34928, 34930-34934, 34936,34942-34944, 34946-34948, 34950-34954, 34958, 34960-34964, 34966-34968,34974-34979, 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61917-61920, 61922, 61925, 61928-61942,61944-61947, 61950-61953, 61955, 61957-61959, 61962, 61966-61969, 61971,61975-61976, 61978-61983, 61987-61988, 61993-61997, 61999-62007, 62009,62011-62013, 62015-62017, 62019, 62024, 62028, 62030-62033, 62035,62038, 62046, 62048-62049, 62051, 62058, 62061-62062, 62069-62076,62078-62082, 62086, 62093-62095, 62097-62102, 62104-62105, 62107-62109,62111, 62114, 62116, 62119-62124, 62126-62130, 62132-62133, 62135,62137, 62140-62145, 62147-62151, 62153-62154, 62160-62166, 62168-62169,62174-62178, 62180, 62184, 62186, 62188, 62191, 62195, 62197-62198,62200, 62202-62208

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

1. A single stranded oligonucleotide having a sequence 5′-X—Y—Z, whereinX is any nucleotide, Y is a nucleotide sequence of 6 nucleotides inlength that is not a seed sequence of a human microRNA, and Z is anucleotide sequence of 1-23 nucleotides in length, wherein the singlestranded oligonucleotide is complementary with at least 8 consecutivenucleotides of a PRC2-associated region of a BDNF gene.
 2. The singlestranded oligonucleotide of claim 1, wherein the oligonucleotide doesnot comprise three or more consecutive guanosine nucleotides.
 3. Thesingle stranded oligonucleotide of claim 1, wherein the oligonucleotidedoes not comprise four or more consecutive guanosine nucleotides.
 4. Thesingle stranded oligonucleotide of claim 1, wherein the oligonucleotideis 8 to 30 nucleotides in length.
 5. The single stranded oligonucleotideof claim 1, wherein the oligonucleotide is 8 to 10 nucleotides in lengthand all but 1, 2, or 3 of the nucleotides of the complementary sequenceof the PRC2-associated region are cytosine or guanosine nucleotides. 6.The single stranded oligonucleotide of claim 1, wherein at least onenucleotide of the oligonucleotide is a nucleotide analogue.
 7. Thesingle stranded oligonucleotide of claim 6, wherein the at least onenucleotide analogue results in an increase in Tm of the oligonucleotidein a range of 1 to 5° C. compared with an oligonucleotide that does nothave the at least one nucleotide analogue.
 8. The single strandedoligonucleotide of claim 1, wherein at least one nucleotide of theoligonucleotide comprises a 2′ O-methyl.
 9. The single strandedoligonucleotide of claim 1, wherein each nucleotide of theoligonucleotide comprises a 2′ O-methyl.
 10. The single strandedoligonucleotide of claim 1, wherein the oligonucleotide comprises atleast one ribonucleotide, at least one deoxyribonucleotide, or at leastone bridged nucleotide.
 11. The single strand oligonucleotide of claim10, wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotideor a ENA modified nucleotide.
 12. The single stranded oligonucleotide ofclaim 1, wherein each nucleotide of the oligonucleotide is a LNAnucleotide.
 13. The single stranded oligonucleotide of claim 1, whereinthe nucleotides of the oligonucleotide comprise alternatingdeoxyribonucleotides and 2′-fluoro-deoxyribonucleotides.
 14. The singlestranded oligonucleotide of claim 1, wherein the nucleotides of theoligonucleotide comprise alternating deoxyribonucleotides and2′-O-methyl nucleotides.
 15. The single stranded oligonucleotide ofclaim 1, wherein the nucleotides of the oligonucleotide comprisealternating deoxyribonucleotides and ENA nucleotide analogues.
 16. Thesingle stranded oligonucleotide of claim 1, wherein the nucleotides ofthe oligonucleotide comprise alternating deoxyribonucleotides and LNAnucleotides.
 17. The single stranded oligonucleotide of claim 13,wherein the 5′ nucleotide of the oligonucleotide is adeoxyribonucleotide.
 18. The single stranded oligonucleotide of claim 1,wherein the nucleotides of the oligonucleotide comprise alternating LNAnucleotides and 2′-O-methyl nucleotides.
 19. The single strandedoligonucleotide of claim 18, wherein the 5′ nucleotide of theoligonucleotide is a LNA nucleotide.
 20. The single strandedoligonucleotide of claim 1, wherein the nucleotides of theoligonucleotide comprise deoxyribonucleotides flanked by at least oneLNA nucleotide on each of the 5′ and 3′ ends of thedeoxyribonucleotides.
 21. The single stranded oligonucleotide of claim1, further comprising phosphorothioate internucleotide linkages betweenat least two nucleotides.
 22. The single stranded oligonucleotide ofclaim 21, further comprising phosphorothioate internucleotide linkagesbetween all nucleotides.
 23. The single stranded oligonucleotide ofclaim 1, wherein the nucleotide at the 3′ position of theoligonucleotide has a 3′ hydroxyl group.
 24. The single strandedoligonucleotide of claim 1, wherein the nucleotide at the 3′ position ofthe oligonucleotide has a 3′ thiophosphate.
 25. The single strandedoligonucleotide of claim 1, further comprising a biotin moietyconjugated to the 5′ nucleotide.
 26. A single stranded oligonucleotidecomprising a region of complementarity that is complementary with atleast 8 consecutive nucleotides of a PRC2-associated region of a BDNFgene, wherein the oligonucleotide has at least one of: a) a sequencethat is 5′X—Y—Z, wherein X is any nucleotide and wherein X is anchoredat the 5′ end of the oligonucleotide, Y is a nucleotide sequence of 6nucleotides in length that is not a human seed sequence of a microRNA,and Z is a nucleotide sequence of 1 to 23 nucleotides in length; b) asequence that does not comprise three or more consecutive guanosinenucleotides; c) a sequence that has less than a threshold level ofsequence identity with every sequence of nucleotides, of equivalentlength to the second nucleotide sequence, that are between 50 kilobasesupstream of a 5′-end of an off-target gene and 50 kilobases downstreamof a 3′-end of the off-target gene; d) a sequence that is complementaryto a PRC2-associated region that encodes an RNA that forms a secondarystructure comprising at least two single stranded loops; and/or e) asequence that has greater than 60% G-C content.
 27. The single strandedoligonucleotide of claim 26, wherein the oligonucleotide has thesequence 5′X—Y—Z and wherein the oligonucleotide is 8-50 nucleotides inlength.
 28. A composition comprising a single stranded oligonucleotideof claim 1 and a carrier.
 29. A composition comprising a single strandedoligonucleotide of claim 1 in a buffered solution.
 30. A composition ofclaim 28, wherein the oligonucleotide is conjugated to the carrier. 31.The composition of claim 30, wherein the carrier is a peptide.
 32. Thecomposition of claim 30, wherein the carrier is a steroid.
 33. Apharmaceutical composition comprising a composition of claim 28 and apharmaceutically acceptable carrier.
 34. A kit comprising a containerhousing the composition of claim
 28. 35. A method of increasingexpression of a BDNF gene in a cell, the method comprising deliveringthe single stranded oligonucleotide of claim 1 into the cell.
 36. Themethod of claim 35, wherein delivery of the single strandedoligonucleotide into the cell results in a level of expression of theBDNF gene that is at least 50% greater than a level of expression of theBDNF gene in a control cell that does not comprise the single strandedoligonucleotide.
 37. A method increasing levels of a BDNF gene in asubject, the method comprising administering the single strandedoligonucleotide of claim 1 to the subject.
 38. A method of treating acondition associated with decreased levels of a BDNF gene in a subject,the method comprising administering the single stranded oligonucleotideof claim 1 to the subject.
 39. The method of claim 38, wherein thecondition is a neurodegeneration disease, such as amyotrophic lateralsclerosis (ALS, also known as Lou Gehrig's disease), Alzheimer's Disease(AD), and Parkinson's Disease (PD).